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Article

Development of a New LC-MS/MS Screening Method for Detection of 120 NPS and 43 Drugs in Blood

by
Fabio Vaiano
1,2,*,
Elisabetta Bertol
2,
Maria Mineo
1,
Laura Pietrosemoli
1,
Jolanda Rubicondo
1,
Claudiu T. Supuran
2,3 and
Fabrizio Carta
2,3
1
Forensic Toxicology Division, Department of Health Science, University of Florence, 50121 Florence, Italy
2
U.R.I.To.N—Unit of Research, Department of Health Science, University of Florence, 50121 Florence, Italy
3
Sezione di Scienze Farmaceutiche e Nutraceutiche, NEUROFARBA Department, University of Florence, 50121 Florence, Italy
*
Author to whom correspondence should be addressed.
Separations 2021, 8(11), 221; https://0-doi-org.brum.beds.ac.uk/10.3390/separations8110221
Submission received: 18 October 2021 / Revised: 9 November 2021 / Accepted: 15 November 2021 / Published: 17 November 2021
(This article belongs to the Section Forensics/Toxins)
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Abstract

:
In the last few years, liquid chromatography coupled with mass spectrometry (LC/MS) has been increasingly used for screening purposes in forensic toxicology. These techniques have the advantages of low time/resource-consuming and high versatility and have been applied in numerous new multi-analytes methods. The new psychoactive substance (NPS) phenomenon provided a great impulse to this wide-range approach, but it is also important to keep the attention on “classical” psychoactive substances, such as benzodiazepines (BDZ). In this paper, a fully validated screening method in blood for the simultaneous detection of 163 substances (120 NPS and 43 other drugs) by a dynamic multiple reaction monitoring analysis through LC-MS/MS is described. The method consists of a deproteinization of 200 µL of blood with acetonitrile. The LC separation is achieved with a 100 mm long C18 column in 35 min. The method was very sensitive, with limits of quantification from 0.02 to 1.5 ng/mL. Matrix effects did not negatively affect the analytical sensitivity. This method proved to be reliable and was successfully applied to our routinary analytical activity in several forensic caseworks, allowing the identification and quantification of many BDZs and 3,4-methylenedioxypyrovalerone (MDPV).

1. Introduction

The increasing number and variety of substances of interest for Forensic Toxicology has required the development of new multi-analyte detection methods. These procedures are very effective tools for identifying and quantifying larger ranges of compounds through single sample extractions, with low time and resource consumption. Besides the traditional screening tests (i.e., immunoassays), which are affected by low specificity and sensitivity, gas chromatography-mass spectrometry (GC-MS) systems are the broadest used techniques for general unknown analyses [1,2,3]. Indeed, electron-impact full scan acquisition allows to compare the mass spectra of unknown compounds with almost “unlimited” mass spectral libraries, always updated and highly reproducible. However, GC-MS analyses are limited to thermostable compounds and require longer sample treatment procedures that often involve derivatization steps [4].
In recent years, these drawbacks of GC-MS have been overcome by the liquid chromatography with tandem MS (LC-MS/MS). Indeed, LC-MS/MS has proven to be less demanding than GC-MS for sample preparation, which can consist in a liquid–liquid extraction (LLE), in a protein precipitation (PP) or even in a dilution and direct injection without the need for removal of the aqueous phase [5,6,7,8]. The derivatization step is not required even if it can be performed to improve the ionization efficiency (IE) [9,10,11]. The low IE of some compounds and unavailability of mass spectra libraries are the main issues concerning LC-MS/MS applications. However, high-resolution MS (HRMS) expanded the breadth of LC-MS applications to even non-targeted analyses, allowing the achievement of general unknown screening analyses beyond the GC-MS [12,13,14,15].
The new psychoactive substance (NPS) phenomenon has represented a great boost to these new analytical approaches [16]. According to latest European Monitoring Centre for Drugs and Drug Addiction (EMCDDA) report [17], 830 compounds are currently being monitored, and new molecules are still emerging (46 in 2020). In Italy, the most prevalent new psychoactive substances (NPSs) are the synthetic cathinones (mephedrone, α-PHP, 3-MMC, eutylone), synthetic cannabinoids (JWH-122 and JWH-210) and opioids (ocfentanil, 2-methyl-AP-237 and carfentanyl) [18]. Besides the huge number of substances, the great chemical variability and the lack of reference standard materials make their detection an ongoing challenge for forensic toxicologists worldwide and often requires a multidisciplinary approach [19,20]. This great interest has led to the development and validation of a great number of new multi-analytes LC-MS/MS screening methods mainly focused on NPS [6,21,22,23,24,25,26,27].
The aim of this paper was to extend this analytical strategy also to other compounds of forensic toxicological interest, in particular benzodiazepines (BDZs) and other antidepressants.
BDZs are the most prescribed tranquilizers/antidepressants and are considered to be safe. However, they can cause serious impairments that make it hard to carry out many activities, such as driving a car [28,29,30,31]. BDZs are often co-consumed with other psychoactive substances, such us alcohol, that intensify their side effects (i.e., respiratory depression). Moreover, it is well documented that they are used to contrast the insomnia by cocaine and other stimulants, such as amphetamines, MDMA and synthetic cathinones [32,33]. In addition, new “designer BDZs” are available on the illicit drugs’ market, establishing a new class of NPSs. For these reasons, the presence of BDZs and antidepressants in biological matrices, together with the main drugs of abuse and NPSs, is worth investigating in a wide variety of forensic caseworks (i.e., driving under the influence, post-mortem analysis, acute intoxication cases). Because of their thermolability and polarity, which require extensive sample preparations for GC-MS detection, LC-MS/MS is the technique of choice for the detection of BDZs [34,35,36].
The new method here described could represent an important tool for a multi-analyte approach to forensic analytics that aims to reduce time and resource-consuming while keeping the high specificity and sensitivity of LC-MS/MS.

2. Materials and Methods

2.1. Chemicals and Reagents

Acetonitrile (ACN) for the PP step was purchased from Panreac Quimica S.L.U. (Castellar del Vallès, Spain). H2O and ACN for LC-MS/MS were acquired from Biosolve Chimie SARL (Dieuze, France). Formic acid was obtained from Merck KGaA (Darmstadt, Germany). 3-OH-flunitrazepam, 7-aminoclonazepam, 7-aminoflunitrazepam, 7-aminonitrazepam, alprazolam, amitriptyline, bromazepam, brotizolam, citalopram, clonazepam, chlordiazepoxide, delorazepam, diazepam, duloxetine, fentanyl, flunitrazepam, flurazepam, fluoxetine, halazepam (internal standard, IS), levomepromazine, lorazepam, lormetazepam, midazolam, mirtazapine, nordiazepam, oxazepam, oxcarbazepine, paroxetine, pinazepam, prazepam, promazine, quetiapine, temazepam, trazodone, triazolam, zolpidem, zopiclone, α-OH-alprazolam and α-OH-midazolam were purchased from Lipomed Inc. (Cambridge, MA, USA). Amphetamine, methamphetamine, MDA, MDMA, MDEA, ketamine and norketamine were obtained from Chemical Research 2000 s.r.l. (Rome, Italy). (±)-cis-3-methyl-norfentanyl, (±)-trans-3-methyl-norfentanyl, 2F-deschloroketamine, 2-methyl-AP-237, 3,4-MD-α-PHP, 3-MeO-PCE, 5-APB, 5-MAPB, 5Cl-AB-PINACA, 5Cl-THJ01C8, 5F-AKB48, 5F-APP-PICA, 5F-APP-PINACA, 5F-Cumyl-P7AICA, 5F-Cumyl-PeGACLONE, 5F-Cumyl-PINACA, 5F-MDMB-7-PAICA, 5F-NNEI-2′-naphtyl-isomer, 5F-MDMB-PICA, 6-APB, 6-MAPB, AB-CHMINACA, acetyl-fentanyl, acetyl-norfentanyl, ADB-FUBINACA, alfentanil, AP-237, APP-FUBINACA, α-PHP, bentazepam, butyryl-fentanyl, butyryl-norfentanyl, carfentanyl, cinazepam, clonazolam, cumyl-PeGACLONE, cyclopropyl-fentanyl, deschloro-N-et-ketamine, diclazepam, etizolam, eutylone, flualprazolam, furanyl-norfentanyl, furanyl-fentanyl, isobutyryl-fentanyl, isotonitazene, MMB-2201, MDMB-CHMICA, MeOAc-fentanyl, MeOAc-norfentanyl, N-ethyl-pentylone, norfentanyl, ocfentanil, pF-furanyl-fentanyl, ritalinic acid, UR-144 were purchased from Comedical s.r.l. (Trento, Italy) by the Italian Early Warning System and donated to our laboratory. 1-naphyrone, 25D-NBOMe, 25H-NBOMe, 2-AI, 2C-E, 2C-N, 2F-methcathinone, 3,4-dimethyl-methcathinone (3,4-DMMC), 3-MeO-PCP, 3-methyl-methcathinone (3-MMC), 4F-amphetamine (4-FA), 4F-methcathinone, 4-MeO-PCP, 4-methyl-ethcathinone, 4-OH-DiPT, 5F-APINACA, 5-IAI, 5-MeOH-DiPT, AB-FUBINACA, ADB-PINACA, AM-2201, AM-2233, AM-694, buphedrone, butylone, BZP, CB-13, dimethylcathinone, ethcathinone, ethylone, JWH-007, JWH-016, JWH-018, JWH-019, JWH-073, JWH-081, JWH-098, JWH-122, JWH-147, JWH-200, JWH-203, JWH-210, JWH-210-d9 (IS), JWH-250, JWH-251, JWH-302, JWH-307, JWH-398, m-CPP, MDAI, MDPV, mephedrone, mephedrone-d3 (IS), methcathinone, methedrone, methoxetamine, methylone, naphyrone, pentedrone, pentylone, pravadoline, RCS-4, RCS-8, WIN 55,212-2 were purchased from LGC standards (Milan, Italy). All standards were diluted to the appropriate concentration with MeOH. Blank blood samples were collected from laboratory personnel and volu78nteers non-consumers of any drug.

2.2. Sample Treatment

Sample treatment was based on a previously published procedure with minimal improvement to increase the efficiency of the PP step [37]. Briefly, 200 µL of blood was added with 700 µL of cold ACN (0 °C) and 10 µL of IS solutions (1 ng/µL). After vortex mixing and centrifugation (2500 G, 5 min), the supernatant was dried under a nitrogen stream at 40 °C and reconstituted with 100 µL of H2O for LC-MS/MS.

2.3. LC-MS/MS

MS analysis was conducted using an HPLC Agilent 1290 Infinity system (Agilent Technologies, Palo Alto, CA, USA) interfaced with an Agilent 6460 Triple Quad MS (Agilent Technologies), equipped with an electrospray ion source (ESI) operating in positive mode. The ESI configuration was: gas temperature 325 °C; gas flow rate 10 L/min; nebulizer 20 psi; capillary 4000 V. Multiple reaction monitoring (MRM) transitions (Table 1), data acquisition and elaboration were performed using the Agilent MassHunter Workstation software package. Chromatographic separation was performed through a Zorbax Eclipse Plus C18 (2.1 × 100 mm, 1.8 µm, Agilent Technologies). The mobile phase initially consisted of 5 mM aqueous formic acid (A) and ACN (B) 99:1. The gradient of elution was carried out as follows: from 0–5 min, linear ramp from 0–5%B; from 5–7 min, ramp to 10%B; isocratic hold from 7 to 10 min; from 10–15 min, ramp to 20%B; from 15–20 min, ramp to 30%B; isocratic hold up to 22 min; form 22 to 25 min, ramp to 40%B; from 25 to 28 min, ramp to 50%B; from 28 to 30 min, ramp to 70%B; from 30 to 35 min to 100%B and isocratic hold to 37 min. Post-time was set at 2 min. The flow rate was 0.6 mL/min.

2.4. Validation Parameters

2.4.1. Selectivity and Specificity

Potential endogenous interfering peaks were evaluated by the measurement of 10 different blank whole blood samples. Exogenous interferences were estimated by spiking 10 different blank blood samples with 500 ng/mL of common drugs and their metabolites (including barbiturates, cannabinoids, cocaine, opioids, etc.).

2.4.2. Sensitivity

The limit of detection (LOD) and the limit of quantification (LOQ) were the lower concentrations that met the identification, precision and accuracy criteria at signal-to-noise ratios (SNR) ≥3 and ≥10, respectively [38]. The evaluation was performed for three replicates of blank specimens fortified with decreasing quantities of each substance.

2.4.3. Linearity, Accuracy and Precision

Due to the low concentration of many available certified standards, we were not able to set up the same calibration curve for all the compounds. For most of them, an eight-point calibration curve from 1 to 500 ng/mL (1, 5, 10, 20, 50, 100, 200, 500 ng/mL) was adopted. For 5-APB, 5Cl-AB-PINACA, 5Cl-THJ-018, 5F-AKB48, 5F-APP-PICA, 5F-APP-PINACA, 5F-cumyl-PINACA, 5-MAPB, 6-APB, 6-MAPB, AB-CHMINACA, acetyl-fentanyl, ADB-FUBINACA, alfentanil, APP-FUBINACA, cumyl-PeGACLONE, MeOAc-norfentanyl, MMB2201 and ritalinic acid, the range was 1–200 ng/mL (1, 5, 10, 20, 50, 100, 200 ng/mL), while for (±)-cis-3-methyl-fentanyl, (±)-trans-3-methyl-fentanyl, butyryl-norfentanyl, furanyl-norfentanyl and norfentanyl, it was 1–100 ng/mL (1, 5, 10, 20, 50, 100 ng/mL). Five replicates of blank blood spiked at the proper concentrations were analyzed, and the least-squares regression procedure was applied to the data. Four quality control (QC) samples were prepared by spiking blank blood at concentration levels of 1, 15, 50 and 75 or 150 or 250 ng/mL depending on the relative calibration curve.
Accuracies and precisions were assessed by analyzing five replicates of each QC sample. Accuracy was expressed as the % mean relative error (%MRE) and the precision as the average of the relative standard deviation (%RSD). Inter-day precisions were established on the basis of 5 analyses performed over the course of one month.

2.4.4. Relative Recovery (RR), Matrix Effect (ME), Stability and Carry over

The estimation of RRs was achieved by the comparison of analytes’ area from QCs prepared before and after the extraction. MEs were calculated comparing the slopes from spiked water solutions and spiked blank blood samples at QC concentrations for three replicates. The stabilities were evaluated by comparing the quantitative results obtained from five replicates of freshly fortified samples (at QC concentration levels) with those obtained from five replicates of the same sample stored at −25 °C and thawed weekly for a month. Carry over estimation was achieved by injecting the extracted blank samples into the LC-MS/MS system immediately after the highest calibrator over five runs.

3. Results and Discussion

3.1. MRM Transitions and Chromatographic Separation

MRM transitions for each compound were obtained through the Agilent Mass Hunter Optimizer, and the two most intense were used as the quantifier and qualifier (Table 1). The method includes 332 transitions. Due to the high number of compounds, acquisition was by dynamic MRM (retention time window: 1 min; max concurrent MRM: 30; dwell range: 13.17–246.50 ms). Chromatographic separation was achieved by a 100 mm long C18 column. Various gradients of elution were tested in order to obtain the best chromatographic performances in term of peaks’ shapes, co-eluting compounds and isomeric pairs resolution. The final chromatographic run was 35 min long (Figure 1, Figure 2 and Figure 3), with a retention range from 1.9 (BZP) to 33.9 min (CB-13). Unfortunately, co-elution was not completely removed even if it did not negatively affect the sensitivity and quantification of the involved substances. The isomeric pairs 1-naphyrone/naphyrone (Figure 1, #34 and #35), (±)-cis-3-met-norfentanyl/(±)-trans-3-met-norfentanyl (Figure 1, #40 and #41) and isobutyryl-fentanyl/butyryl-fentanyl (Figure 1, #54 and #55) were chromatographically baseline-separated. The separation of these opioids’ couples represents a great advantage of this method since, to the best of our knowledge, no peer reviewed literature reported their chromatographic resolution to the baseline [26]. 3-MeO-PCP and 4-MeO-PCP (Figure 1, #73 and #74) had an identical retention time (16.7 min), but the effective identification can be achieved by their quantifier transitions—189 m/z for 3-MeO-PCP and 86 m/z for 4-MeO-PCP [39]. The m/z ratio can be useful for the couple 2F-methcathione and 4F-methcathinone (Figure 1, #16 and #17), whose quantifier/qualifier ratios are 2.9 and 1.1, respectively. However, this approach cannot be applied if both the molecules are present in the same sample, as the shared product ions modify the measured ratio. For the isomeric pairs 3-MMC/mephedrone (Figure 1, #25 and #26), 5/6-APB (Figure 1, #7 and #8) and 5/6-MAPB (Figure 1, #9 and #10), discrimination cannot be achieved by the MRM transitions, and the chromatographic gradient did not allow a full baseline resolution. However, when individually analyzed, these compounds can be distinguished based on the retention time.
All the other compounds sharing the [M+H]+ ion and/or the MRM transitions were chromatographically identifiable.

3.2. Method Validation

The sample treatment procedure was optimized starting from two methods currently used in our laboratory for NPS and BDZ/antidepressants quantification. The first one consists of a protein precipitation with ACN, while the second procedure requires an LLE with an 8:2 mixture of dichloromethane/ethyl-acetate at pH 4.5 (phosphate buffer). Both methods were preliminarily tested at QC levels, and the evaluation was based on RR (>75%), ME (from −30 to +30%) and time/resource-consuming. LLE provided the best outcomes for BDZ/antidepressants (RR > 90%; ME from −5 to +7%) but was not suitable for most NPSs, mainly due to the low RRs (i.e., for synthetic cannabinoids, the RR was from 50–70%). On the contrary, PP resulted as very versatile with acceptable criteria met by all the compounds; moreover, it is even faster and simpler. Thus, this treatment was chosen for the new procedure and subsequently optimized at varying volumes (from 500 to 1000 µL) and temperatures (from −25 to 0 °C) of ACN. The optimal conditions have been reported above. Compared to published procedures, which require extraction phases (LLE or solid-phase extraction), our method seemed to be simpler and faster [6,21,22,23,25,26,27].
The method proved to be highly specific and selective since no endogenous and exogenous interfering peaks were observed. The coefficient of determination (R2) was always above 0.9900 in all three of the different tested ranges.
Sensitivity was in line with previously published methods. LOQ ranged from 0.02 ng/mL for prazepam to 1.5 ng/mL for 2F-methcathinone (Table 2). Regarding the main classes of substances, we can state that the highest sensitivities were observed among the BDZs with values always <0.5 ng/mL, except for the “designer BDZ” cinazepam and the metabolites oxazepam and 7-aminoclonazepam (1 ng/mL). Low LOQ levels were also registered for synthetic cannabinoids (0.05–0.5 ng/mL, except for ADB-FUBINACA, APP-FUBINACA and JWH-007 at 1 ng/mL) and fentanyl analogues (0.1–0.5 ng/mL, except for acetyl-norfentanyl, alfentanil and norfentanyl at 1 ng/mL). The method is adequately sensitive to detect all the included substances at recreational or sub-recreational blood concentrations [7,40,41,42]. At the lowest QC level, 39 substances did not meet the acceptance criteria for accuracy (−20% < %MRE < 20%) with the highest value at 22.7% (cumyl-PeGACLONE). Accuracy improved at higher QC concentrations in the following ranges: −20.9%–+21.7% at QC2 (with 16 substances over the acceptance criteria, −20% < %MRE < 20%); −15.0%–+16.9% at QC3 (with 7 substances over the acceptance criteria, −15% < %MRE < 15%); −9.9%–+9.8% at QC4 (with all substances within the acceptance criteria, 10% < %MRE < 10%). In many cases, the lowest QCs corresponded to the LOQ value or were close to it, and this may explain their poor accuracy. Thus, quantification at these low concentrations should be treated with caution. None of the compounds showed poor accuracy across all concentrations. Intra-day and inter-day ranged from 1.0 to 20.0% and 2.0 to 20.9%, respectively.
As described above, ME and RR were not negatively affected by the simple and rapid PP step. The mean ME was 3.5%, with the highest values of ion suppression and ion enhancement at −31.1% (5-MeO-DiPT) and +27.7% (methcathinone), respectively. RR was always >75% and ranged from 75.3% to (4-FA) and 99.0% (5-F-APP-PINACA). Stability studies showed that storage at −25°C and the implementation of freeze/thaw cycles led to a loss <12.8%. Carry over was not observed.

3.3. Application to Real Samples

Before validating this new method, BDZ/antidepressants and NPSs were quantified by means of two separated procedures from two aliquots of blood, entailing double time, resource and sample consuming [43]. The application of this new method significantly improved our analytical activity, especially when a wide-range detection strategies are required. In the last few months, it has been successfully applied in cases of acute intoxications (n = 2), post-mortem analysis (n = 1) and the evaluation of driving under the influence (DUID) for drivers involved in road accidents (n = 48). In DUID cases, the most detected compounds were BDZs: diazepam and its main metabolites nordiazepam, temazepam and oxazepam (n = 5), alprazolam (n = 3) and lorazepam (n = 2), and they were always within or below the therapeutic concentrations (Table 3) [40]. Midazolam, fentanyl and ketamine were also detected when administrated by the healthcare personnel at the emergency department to induce sedation. Moreover, ketamine and its main metabolite (norketamine) were also quantified in two DUID cases (Figure 4). MDPV was found at 42.3 ng/mL in a blood sample from a 23-year-old man who was hospitalized due to severe agitation. None of the newest NPSs was detected.

4. Conclusions

An LC-MS/MS-based multi-analyte method was fully validated. The here described procedure employs a simple sample preparation (PP) and requires a small sample volume (200 µL of blood) for the detection of 120 NPS and 43 drugs of great forensic-toxicological interest, such as BDZs. The short preparation time represents this analytical method’s great advantage, as well as its high chromatographic efficiency. Moreover, the high specificity and sensitivity make this methodology suitable for all cases that require the identification and quantification of a wide range of compounds. Implementation in our routinary activity was actual proof of the advantages of this method, as well as its efficacy and reliability. In the future, the new method will be used to study the prevalence of BDZ/antidepressants in road accidents.

Author Contributions

Conceptualization, F.V. and F.C.; methodology, F.V.; software, F.V.; validation, F.V. and F.C.; formal analysis, F.V., M.M., L.P. and J.R.; investigation, F.V., M.M., L.P. and J.R.; resources, F.V.; data curation, F.V. and M.M.; writing—original draft preparation, F.V. and F.C.; writing—review and editing, F.V. and F.C.; visualization, C.T.S. and E.B.; supervision, C.T.S. and E.B.; All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Informed consent was obtained from all subjects involved in the study.

Conflicts of Interest

The authors declare no conflict of interest.

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Separations 08 00221 g001 550
Figure 1. Chromatograms for the quantifier MRM transitions of phenethylamine, synthetic cathinones, fentanyl analogues and unclassified compounds, with a retention time within 0–20 min at 5 ng/mL. 1. MDEA; 2. amphetamine; 3. 4-FA; 4. methamphetamine; 5. MDMA; 6. 2C-N; 7. 5-APB; 8. 6-APB; 9. 5-MAPB; 10. 6-MAPB; 11. MDA; 12. 2C-E 13. 25H-NBOMe; 14. 25D-NBOMe; 15. methcathinone; 16. 2F-methcathinone; 17. 4F-methcathinone; 18. dimethylcathinone; 19. methylone; 20. ethcathinone; 21. ethylone; 22. methedrone; 23. buphedrone; 24. butylone; 25. 3-MMC; 26. Mephedrone; 27. eutylone; 28. 4-methyl-ethcathinone; 29. pentedrone; 30. pentylone; 31. 3,4-DMMC; 32. N-ethylpentylone; 33. MDPV; 34. 1-naphyrone; 35. naphyrone; 36. MeOAc-norfentanyl; 37. acetyl-norfentanyl; 38. norfentanyl; 39. furanyl-norfentanyl; 40. (±)-cis-3-met-norfentanyl; 41. (±)-trans-3-met-norfentanyl; 42. MeOAc-fentanyl; 43. butyryl-norfentanyl; 44. AP-237; 45. 2-methyl-AP-237; 46. acetyl-fentanyl; 47. ocfentanil; 48. alfentanyl; 49. fentanyl; 50. furanyl-fentanyl; 51. cyclopropyl-fentanyl; 52. pF-furanyl-fentanyl; 53. carfentanyl; 54. isobutyryl-fentanyl; 55. butyryl-fentanyl; 56. BZP; 57. 2-AI; 58. Isotonitazene; 59. MDAI; 60. 2F-deschloroketamine; 61. ritalinic acid; 62. deschloro-N-et-ketamine; 63. norketamine; 64. ketamine; 65. 4-OH-DiPT; 66. m-CPP; 67. 5-IAI; 68. methoxetamine; 69. 5-MeO-DiPT; 70. α-PHP; 71. 3,4MD-a-PHP; 72. 3-MeO-PCE; 73. 3-MeO-PCP; 74. 4-MeO-PCP.
Figure 1. Chromatograms for the quantifier MRM transitions of phenethylamine, synthetic cathinones, fentanyl analogues and unclassified compounds, with a retention time within 0–20 min at 5 ng/mL. 1. MDEA; 2. amphetamine; 3. 4-FA; 4. methamphetamine; 5. MDMA; 6. 2C-N; 7. 5-APB; 8. 6-APB; 9. 5-MAPB; 10. 6-MAPB; 11. MDA; 12. 2C-E 13. 25H-NBOMe; 14. 25D-NBOMe; 15. methcathinone; 16. 2F-methcathinone; 17. 4F-methcathinone; 18. dimethylcathinone; 19. methylone; 20. ethcathinone; 21. ethylone; 22. methedrone; 23. buphedrone; 24. butylone; 25. 3-MMC; 26. Mephedrone; 27. eutylone; 28. 4-methyl-ethcathinone; 29. pentedrone; 30. pentylone; 31. 3,4-DMMC; 32. N-ethylpentylone; 33. MDPV; 34. 1-naphyrone; 35. naphyrone; 36. MeOAc-norfentanyl; 37. acetyl-norfentanyl; 38. norfentanyl; 39. furanyl-norfentanyl; 40. (±)-cis-3-met-norfentanyl; 41. (±)-trans-3-met-norfentanyl; 42. MeOAc-fentanyl; 43. butyryl-norfentanyl; 44. AP-237; 45. 2-methyl-AP-237; 46. acetyl-fentanyl; 47. ocfentanil; 48. alfentanyl; 49. fentanyl; 50. furanyl-fentanyl; 51. cyclopropyl-fentanyl; 52. pF-furanyl-fentanyl; 53. carfentanyl; 54. isobutyryl-fentanyl; 55. butyryl-fentanyl; 56. BZP; 57. 2-AI; 58. Isotonitazene; 59. MDAI; 60. 2F-deschloroketamine; 61. ritalinic acid; 62. deschloro-N-et-ketamine; 63. norketamine; 64. ketamine; 65. 4-OH-DiPT; 66. m-CPP; 67. 5-IAI; 68. methoxetamine; 69. 5-MeO-DiPT; 70. α-PHP; 71. 3,4MD-a-PHP; 72. 3-MeO-PCE; 73. 3-MeO-PCP; 74. 4-MeO-PCP.
Separations 08 00221 g001
Separations 08 00221 g002 550
Figure 2. Chromatograms for the quantifier MRM transitions of synthetic cannabinoids compounds, with a retention time within 18–34 min at 5 ng/mL. 75. pravadoline; 76. AM-2233; 77. JWH-200; 78. AB-FUBINACA; 79. 5F-APP-PICA; 80. 5F-APP-PINACA; 81. 5Cl-AB-PINACA; 82. ADB-FUBINACA; 83. APP-FUBINACA; 84. 5F-MDMB-7PAICA; 85. 5F-Cumyl-P7AICA; 86. WIN 55,212-2; 87. AB-CHMINACA; 88. ADB-PINACA; 89. MMB-2201; 90. 5F-MDMB-PICA; 91. 5F-Cumyl-PeGACLONE; 92. 5F-Cumyl-PINACA; 93. 5F-NNEI-2-naphtyl-isomer; 94. AM-2201; 95. RCS-4; 96. JWH-302; 97. MDMB-CHMICA; 98. JWH-250; 99. AM-694; 100. cumyl-PeGLACONE; 101. JWH-073; 102. 5F-APINACA; 103. JWH-251; 104. JWH-016; 105. JWH-203; 106. JWH-018; 107. 5Cl-THJ-018; 108. JWH-007; 109. JWH-081; 110. 5F-AKB-48; 111. JWH-307; 112. JWH-098; 113. JWH-122; 114. JWH-019; 115. RCS-8; 116. UR-144; 117. JWH-210; 118. JWH-398; 119. JWH-147; 120. CB-13.
Figure 2. Chromatograms for the quantifier MRM transitions of synthetic cannabinoids compounds, with a retention time within 18–34 min at 5 ng/mL. 75. pravadoline; 76. AM-2233; 77. JWH-200; 78. AB-FUBINACA; 79. 5F-APP-PICA; 80. 5F-APP-PINACA; 81. 5Cl-AB-PINACA; 82. ADB-FUBINACA; 83. APP-FUBINACA; 84. 5F-MDMB-7PAICA; 85. 5F-Cumyl-P7AICA; 86. WIN 55,212-2; 87. AB-CHMINACA; 88. ADB-PINACA; 89. MMB-2201; 90. 5F-MDMB-PICA; 91. 5F-Cumyl-PeGACLONE; 92. 5F-Cumyl-PINACA; 93. 5F-NNEI-2-naphtyl-isomer; 94. AM-2201; 95. RCS-4; 96. JWH-302; 97. MDMB-CHMICA; 98. JWH-250; 99. AM-694; 100. cumyl-PeGLACONE; 101. JWH-073; 102. 5F-APINACA; 103. JWH-251; 104. JWH-016; 105. JWH-203; 106. JWH-018; 107. 5Cl-THJ-018; 108. JWH-007; 109. JWH-081; 110. 5F-AKB-48; 111. JWH-307; 112. JWH-098; 113. JWH-122; 114. JWH-019; 115. RCS-8; 116. UR-144; 117. JWH-210; 118. JWH-398; 119. JWH-147; 120. CB-13.
Separations 08 00221 g002
Separations 08 00221 g003 550
Figure 3. Chromatograms for the quantifier MRM transitions of BDZ/antidepressants, with a retention time within 6–30 min at 5 ng/mL: 121. 7-aminonitrazepam; 122. 7-aminoclonazepam; 123. mirtazapine; 124. 7-aminoflunitrazepam; 125. zolpidem; 126. chlordiazepoxide; 127. bentazepam; 128. oxcarbazepine; 129. midazolam; 130. trazodone; 131. bromazepam; 132. quetiapine; 133. α-OH-midazolam; 134. flurazepam; 135. zoplicone; 136. citalopram; 137. promazine; 138. 3-OH-flunitrazepam; 139. paroxetine; 140. clonazolam; 141. α-OH-alprazolam; 142. oxazepam; 143. duloxetina; 144. amitriptyline; 145. flualprazolam; 146. clonazepam; 147. lorazepam; 148. fluoxetine; 149. alprazolam; 150. nordiazepam; 151. flunitrazepam; 152. triazolam; 153. temazepam; 154. brotizolam; 155. etizolam; 156. delorazepam; 157. lormetazepam; 158. cinazepam; 159. diazepam; 160. levomepromazine; 161. diclazepam; 162. pinazepam; 163. prazepam.
Figure 3. Chromatograms for the quantifier MRM transitions of BDZ/antidepressants, with a retention time within 6–30 min at 5 ng/mL: 121. 7-aminonitrazepam; 122. 7-aminoclonazepam; 123. mirtazapine; 124. 7-aminoflunitrazepam; 125. zolpidem; 126. chlordiazepoxide; 127. bentazepam; 128. oxcarbazepine; 129. midazolam; 130. trazodone; 131. bromazepam; 132. quetiapine; 133. α-OH-midazolam; 134. flurazepam; 135. zoplicone; 136. citalopram; 137. promazine; 138. 3-OH-flunitrazepam; 139. paroxetine; 140. clonazolam; 141. α-OH-alprazolam; 142. oxazepam; 143. duloxetina; 144. amitriptyline; 145. flualprazolam; 146. clonazepam; 147. lorazepam; 148. fluoxetine; 149. alprazolam; 150. nordiazepam; 151. flunitrazepam; 152. triazolam; 153. temazepam; 154. brotizolam; 155. etizolam; 156. delorazepam; 157. lormetazepam; 158. cinazepam; 159. diazepam; 160. levomepromazine; 161. diclazepam; 162. pinazepam; 163. prazepam.
Separations 08 00221 g003
Separations 08 00221 g004 550
Figure 4. Chromatograms for real case #2 and #4.
Figure 4. Chromatograms for real case #2 and #4.
Separations 08 00221 g004
Table
Table 1. MRM transitions of each compound included in the screening method. Quantitative transitions are shown in bold.
Table 1. MRM transitions of each compound included in the screening method. Quantitative transitions are shown in bold.
CompoundMRM
Transitions
(m/z)		Retention
Time
(min)		CompoundMRM
Transitions
(m/z)		Retention
Time
(min)
BDZ/Antidepressantsisobutyryl-fentanyl351: 188, 10518.9
3-OH-flunitrazepam330: 311, 28419.9isotonitazene411: 100, 7219.8
7-aminoclonazepam286: 222, 12110.3MeOAc-fentanyl353: 188, 1056.3
7-aminoflunitrazepam284: 135, 22713.2MeOAc-norfentanyl249: 84, 5515.3
7-aminonitrazepam252: 121, 2086.5norfentanyl233: 84, 559.5
alprazolam309: 281, 20521.9ocfentanil371: 188, 10515.7
amitriptyline278: 91, 10520.9pF-furanyl-fentanyl393: 188, 10518.7
bentazepam297: 166, 26917.3Synthetic Cannabinoids
bromazepam316: 182, 20917.65Cl-AB-PINACA365: 320, 24926.7
brotizolam393: 314, 27923.55Cl-THJ-018377: 249, 14531.9
chlordiazepoxide300: 283, 28215.45F-AKB-48384: 135, 9332.1
cinazepam465: 347, 31924.75F-APINACA384: 135, 9331.3
citalopram325: 109, 26218.55F-APP-PICA396: 232, 37926.4
clonazepam316: 270, 21421.45F-APP-PINACA397: 233, 35226.8
clonazolam354: 308, 28020.45F-Cumyl-P7AICA368: 250, 14528.4
delorazepam305: 165, 140245F-Cumyl-PeGACLONE391: 273, 18530.5
diazepam285: 154, 193255F-Cumyl-PINACA368: 233, 25030.5
diclazepam319: 227, 15426.15F-MDMB-7PAICA378: 318, 14527.8
duloxetina298: 154, 15720.85F-MDMB-PICA377: 232, 14429.7
etizolam343: 314, 28923.85F-NNEI-2-naphtyl-isomer375: 232, 14430.6
flualprazolam327: 292, 29921.3AB-CHMINACA357: 241, 31228.7
flunitrazepam314: 268, 23922.6AB-FUBINACA369: 324, 10926
fluoxetine310: 148, 11721.8ADB-FUBINACA383: 338, 25327.3
flurazepam388: 315, 31718.1ADB-PINACA345: 215, 30028.7
halazepam353: 241, 22228.7AM-2201360: 155, 12730.7
levomepromazine329: 148, 13025.7AM-2233459: 98, 11219.9
lorazepam321: 275, 30321.5AM-694436: 190, 27231.2
lormetazepam335: 289, 31724.4APP-FUBINACA417: 372, 10927.6
midazolam326: 291, 22317.5CB-13369: 155, 12733.9
mirtazapine266: 195, 7212.2cumyl-PeGLACONE373: 255, 18531.2
nordiazepam271: 165, 14022.5JWH-007356: 155, 12732
oxazepam287: 269, 24120.6JWH-016342: 127, 15531.4
oxcarbazepine253: 208, 23617.4JWH-018342: 155, 12731.7
paroxetine330: 70, 19220.1JWH-019356: 155, 12732.4
pinazepam309: 241, 26927.2JWH-073328: 155, 12731.2
prazepam325: 271, 14028.6JWH-081372: 185, 15732
promazine285: 86, 5819.7JWH-098386: 185, 15732.2
quetiapine384: 253, 22117.6JWH-122356: 169, 14132.2
temazepam301: 255, 28323.2JWH-147382: 155, 12732.9
trazodone372: 176, 14817.5JWH-200385: 155, 11421.4
triazolam343: 308, 31522.6JWH-203340: 125, 23831.5
zolpidem308: 235, 23614.4JWH-210370: 183, 15532.7
zoplicone389: 217, 24518.3JWH-210-d9379: 183, 15532.6
α-OH-alprazolam325: 297, 21620.5JWH-250336: 121, 9131.1
α-OH-midazolam342: 168, 20317.7JWH-251320: 105, 14431.4
MiscellaneousJWH-302336: 214, 12130.8
2-AI134: 117, 1153.7JWH-307386: 155, 12732.2
2F-deschloroketamine222: 109, 1637.8JWH-398376: 189, 16132.7
3,4MD-α-PHP290: 135, 14015.6MDMB-CHMICA385: 240, 14431.3
3-MeO-PCP274: 189, 12116.7MMB-2201363: 232, 14428.8
3-MeO-PCE234: 189, 12115.8pravadoline379: 135, 11419
4-MeO-PCP274: 86, 12116.7RCS-4322: 135, 7730.8
4-OH-DiPT261: 160, 11410.4RCS-8376: 121, 9132.4
5-IAI260: 116, 24311.6UR-144312: 125, 5532.5
5-MeO-DiPT275: 114, 17414.1WIN 55,212-2427: 155, 12728.4
BZP177: 91, 651.9Synthetic Cathinones
deschloro-N-et-ketamine218: 91, 1738.81-naphyrone282: 126, 14117.8
ketamine238: 125, 1799.42F-methcathinone182: 164, 1494.8
m-CPP197: 154, 11810.43,4-DMMC192: 174, 15911.7
MDAI178: 161, 1034.93-MMC178: 160, 1457.8
methoxetamine248: 203, 12111.84F-methcathinone182: 164, 1494.8
norketamine224: 125, 2078.84-methyl-ethcathinone192: 174, 1448.8
ritalinic acid220: 84, 568.6buphedrone178: 131, 1606.9
α-PHP246: 140, 9114.6butylone222: 204, 1747.7
Opioidsdimethylcathinone178: 133, 1054.9
(±)-cis-3-met-norfentanyl247: 98, 6911.7ethcathinone178: 160, 1325.6
(±)-trans-3-met-norfentanyl247: 98, 6911.9ethylone222: 204, 1746.7
2-methyl-AP-237287: 117, 11514.8eutylone236: 218, 1888.4
acetyl-fentanyl323: 188, 10515.6MDPV276: 126, 13513.1
acetyl-norfentanyl219: 84, 556.5mephedrone178: 160, 1457.7
alfentanil417: 268, 19717.3mephedrone-d3181: 148, 1637.6
AP-237273: 117, 11513.8methcathinone164: 146, 1313.7
butyryl-fentanyl351: 188, 10519.2methedrone194: 176, 1616.8
butyryl-norfentanyl247: 84, 5513.3methylone208: 160, 1325.4
carfentanyl395: 335, 11318.9naphyrone282: 141, 21118.5
cyclopropyl-fentanyl349: 188, 10518.3N-ethylpentylone250: 202, 23212.2
fentanyl337: 188, 13217.5pentedrone192: 174, 13210
furanyl-fentanyl375: 188, 10518.2pentylone236: 218, 18811.2
furanyl-norfentanyl271: 84, 5510.9
Table
Table 2. Main validation parameters for each compound included in the screening method. N° of replicates: 3 for LOD, LOQ, ME and RR; 5 for linearity, accuracy and precisions.* 250 ng/mL except for: 5-APB, 5Cl-AB-PINACA, 5Cl-THJ-018, 5F-AKB48, 5F-APP-PICA, 5F-APP-PINACA, 5F-cumyl-PINACA, 5-MAPB, 6-APB, 6-MAPB, AB-CHMINACA, acetyl-fentanyl, ADB-FUBINACA, alfentanil, APP-FUBINACA, cumyl-PeGACLONE, MeOAc-norfentanyl, MMB2201 and ritalinic acid, 150 ng/mL; (±)-cis-3-methyl-fentanyl, (±)-trans-3-methyl-fentanyl, butyryl-norfentanyl, furanyl-norfentanyl and norfentanyl, 75 ng/mL.
Table 2. Main validation parameters for each compound included in the screening method. N° of replicates: 3 for LOD, LOQ, ME and RR; 5 for linearity, accuracy and precisions.* 250 ng/mL except for: 5-APB, 5Cl-AB-PINACA, 5Cl-THJ-018, 5F-AKB48, 5F-APP-PICA, 5F-APP-PINACA, 5F-cumyl-PINACA, 5-MAPB, 6-APB, 6-MAPB, AB-CHMINACA, acetyl-fentanyl, ADB-FUBINACA, alfentanil, APP-FUBINACA, cumyl-PeGACLONE, MeOAc-norfentanyl, MMB2201 and ritalinic acid, 150 ng/mL; (±)-cis-3-methyl-fentanyl, (±)-trans-3-methyl-fentanyl, butyryl-norfentanyl, furanyl-norfentanyl and norfentanyl, 75 ng/mL.
CompoundLOD
(ng/mL)		LOQ
(ng/mL)		R2Accuracy (%)Intra-Day Precision (%)Inter-Day Precision (%)ME
(%)		RR
(%)
Q1Q2Q3Q4 *Q1Q2Q3Q4Q1Q2Q3Q4
(±)-cis-3-met-norfentanyl0.020.10.9949−20.6−14.813.5−2.618.815.714.010.019.211.012.85.6−0.478.4
(±)-trans-3-met-norfentanyl0.010.10.9923−19.7−18.15.0−7.911.017.211.79.410.816.78.13.7−20.194.3
1-naphyrone0.10.50.999220.719.6−14.3−7.919.614.012.01.011.811.49.56.6−19.698.5
25D-NBOMe0.010.050.9997−16.510.75.5−7.314.213.212.15.120.517.07.29.916.886.6
25H-NBOMe0.030.10.9984−15.1−19.014.0−5.719.719.55.69.918.318.514.510.716.290.5
2-AI0.050.10.9909−16.119.6−14.8−8.44.713.76.43.619.617.810.06.3−2.283.6
2C-E0.30.50.998720.8−14.8−14.8−9.95.119.210.05.313.920.013.210.315.685.6
2C-N0.20.50.998813.819.415.1−3.513.719.014.33.819.814.310.94.811.690.6
2F-deschloroketamine0.510.9975−19.7−17.54.1−7.419.913.59.49.910.917.46.47.7−5.398.0
2F-methcathinone0.51.50.996111.021.2−10.9−2.39.719.22.41.319.018.812.18.1−14.789.5
2-methyl-AP-2370.10.50.9981−9.515.52.2−8.812.219.111.19.512.719.56.23.7−14.187.4
3.4-DMMC0.10.30.9973−15.00.115.9−7.619.520.09.93.719.615.915.610.7−14.279.8
3.4MD-α-PHP0.20.50.999721.419.0−10.0−7.519.314.715.06.018.319.115.67.5−20.478.8
3-OH-flunitrazepam0.010.050.9989−11.611.2−0.5−1.83.95.114.78.118.115.010.54.218.198.8
3−MeO-PCP0.030.10.9918−20.319.2−1.12.619.95.26.110.019.610.99.310.0−3.292.6
3-MeO-PCE0.20.50.999319.0−11.712.57.67.615.214.69.511.510.39.56.8−6.897.8
3-MMC0.510.994717.8−20.3−8.38.111.119.714.010.017.311.815.75.7-1.883.0
4F-amphetamine0.10.50.992220.612.3−0.6−2.45.719.66.110.011.016.915.68.310.375.3
4F-methcathinone0.10.30.998914.512.84.5−4.019.819.413.41.519.310.013.33.0−18.898.8
4-MeO-PCP0.10.50.995019.6−14.514.81.76.218.26.09.315.312.011.49.111.392.7
4-methyl-ethcathinone0.10.30.9992−11.114.815.3−5.019.84.514.89.513.712.47.39.1−6.780.9
4-OH-DiPT0.020.20.996615.917.0−12.3−4.715.019.414.19.417.614.510.89.8−16.480.2
5-APB0.20.50.999210.2−20.511.0−9.07.419.214.55.814.113.712.22.2−17.696.5
5Cl-AB-PINACA0.20.50.9932−20.4−17.1−11.3−7.319.019.65.79.819.014.95.97.7−9.776.3
5Cl-THJ-0180.20.50.9969−14.016.4−14.2−9.76.25.714.71.714.213.76.72.110.077.2
5F-AKB-480.050.20.999821.3−11.114.5−8.013.419.05.410.012.919.915.22.0−3.678.5
5F-APINACA0.050.10.991821.020.210.06.512.117.813.29.914.314.515.47.9−17.590.9
5F-APP-PICA0.20.50.998318.621.0−14.09.719.42.49.710.018.917.05.32.020.487.4
5F-APP-PINACA0.10.50.991822.2−13.014.6−5.12.211.514.31.616.017.98.65.9−2.499.0
5F-Cumyl-P7AICA0.20.50.99990.213.6−3.5−7.113.15.914.19.718.112.96.89.719.494.3
5F-Cumyl-PeGACLONE0.050.20.999113.8−17.5−14.15.716.811.112.19.912.215.98.12.14.977.9
5F-Cumyl-PINACA0.050.20.997921.816.6−12.8−8.514.019.714.49.516.219.910.25.8−20.190.3
5F-MDMB-7PAICA0.20.50.999119.5−15.1−10.2−3.13.419.014.37.011.015.114.18.38.477.5
5F-MDMB-PICA0.050.20.998517.312.6−11.9−9.113.410.014.97.315.515.813.83.33.993.9
5F-NNEI-2′-naphtyl-isomer0.10.50.9998−20.218.3−11.7−1.719.219.014.95.510.514.88.38.013.182.1
5-IAI0.110.994512.3−11.5−11.12.11.917.014.83.119.211.214.54.07.081.1
5-MAPB0.510.999012.1−9.6−10.57.29.76.715.04.020.611.26.78.321.094.1
5-MeO-DiPT0.10.30.9939−20.1−12.2−4.1−8.817.78.58.41.413.819.95.22.2−31.180.2
6-APB0.210.998921.815.0−14.2−5.813.24.61.610.012.016.710.64.2−2.588.0
6-MAPB0.510.999921.114.314.79.519.014.63.810.014.520.214.07.0−4.995.8
7-aminoclonazepam0.510.996015.518.17.6−4.47.019.28.79.011.513.16.14.33.577.2
7-aminoflunitrazepam0.010.050.995017.00.53.89.417.99.26.59.916.214.314.99.5−15.896.9
7-aminonitrazepam0.050.20.9967−12.119.314.6−3.25.619.56.51.720.817.915.85.47.678.8
AB-CHMINACA0.020.10.998520.019.25.9−8.34.22.93.410.019.315.614.06.614.388.5
AB-FUBINACA0.10.50.9941−15.319.1−2.0−9.97.81.714.16.118.718.514.53.56.085.2
acetyl-fentanyl0.10.50.999021.119.2−11.8−8.019.114.214.19.615.319.29.59.2−0.776.0
acetyl-norfentanyl0.510.9969−10.7−16.514.8−7.915.019.49.89.015.017.110.09.712.278.2
ADB-FUBINACA0.510.9927−18.0−20.410.7−9.017.120.09.68.814.711.19.68.2−8.392.7
ADB-PINACA0.10.50.999114.519.0−11.40.913.517.23.32.719.412.715.93.71.587.6
alfentanil0.210.9966−14.2−12.28.0−6.418.15.213.09.112.510.35.29.5−0.993.9
alprazolam0.050.10.999612.121.4−13.9−9.814.918.23.69.920.920.36.83.6−20.176.4
AM-22010.050.10.997918.313.2−1.3−6.219.219.712.58.019.720.414.97.515.893.9
AM-22330.050.10.999720.0−16.74.95.019.215.214.45.119.620.912.06.919.382.4
AM-6940.10.50.9986−14.80.32.7−7.512.94.75.89.919.015.47.12.615.191.1
amitriptyline0.510.9933−20.619.80.7−5.019.019.21.49.013.820.25.94.2−2.880.5
amphetamine0.10.50.9941−18.0−20.9−13.9−6.33.611.29.94.316.010.58.45.215.477.9
AP-2370.050.10.9955−21.415.312.2−3.03.512.56.09.912.819.713.810.5−2.595.4
APP-FUBINACA0.510.9978−13.619.53.5−2.07.711.82.82.619.311.27.43.6−19.877.0
bentazepam0.20.50.998918.2−16.8−5.19.519.08.63.99.814.117.26.68.311.190.6
bromazepam0.20.50.995920.3−17.2−14.6−7.91.818.214.99.019.119.813.88.020.491.9
brotizolam0.030.10.99821.0−15.6−8.6−8.011.76.43.89.119.520.95.32.220.177.1
buphedrone0.10.50.995915.919.0−13.50.16.419.814.410.020.213.714.77.912.384.4
butylone0.10.30.997319.813.512.29.519.68.214.92.319.113.710.32.8−3.086.4
butyryl-fentanyl0.10.50.996711.5−16.57.0−7.02.118.58.89.915.111.214.72.89.998.5
butyryl-norfentanyl0.10.50.997021.6−14.0−1.6−5.319.02.52.810.010.220.214.98.812.792.2
BZP0.050.50.9989−21.9−17.2−14.6−5.49.912.62.66.016.520.313.38.915.590.5
carfentanyl0.10.50.995211.412.2−11.5−9.819.85.814.79.916.311.411.910.4−9.587.0
CB-130.10.50.9983−10.1−11.513.0−8.47.49.714.49.814.914.59.04.510.994.0
chlordiazepoxide0.20.50.999612.7−17.914.99.712.915.714.52.319.719.414.65.2−0.791.6
cinazepam0.510.9984−20.4−11.8−0.6−3.419.019.74.19.619.613.99.65.814.288.9
citalopram0.030.10.996919.621.7−13.3−7.419.019.514.09.813.614.36.410.4−6.794.5
clonazepam0.10.50.998119.619.3−9.1−6.619.71.813.55.214.118.615.03.68.086.4
clonazolam0.20.50.9987−9.417.1−15.0−6.419.04.114.12.716.415.514.56.25.981.3
cumyl-PeGLACONE0.20.50.992722.7−18.23.68.01.719.214.49.516.616.414.96.616.295.4
cyclopropyl-fentanyl0.10.50.9988−17.2−16.0−14.26.517.219.814.59.319.620.18.03.26.980.9
delorazepam0.10.50.999619.911.116.9−4.71.219.24.49.610.112.09.39.3−18.881.6
deschloro-N-et-ketamine0.20.50.9958−19.3−11.511.1−3.519.019.79.39.920.113.56.05.5−7.396.0
diazepam0.010.050.998015.916.812.3−7.97.112.44.19.712.220.714.42.620.578.3
diclazepam0.20.50.990815.1−20.75.4−2.019.618.614.17.610.819.89.98.720.298.9
dimethylcathinone0.10.30.9987−18.5−20.4−9.95.58.213.09.59.819.120.99.19.9−14.493.9
duloxetina0.10.50.997611.3−13.87.27.419.82.95.52.410.013.27.73.0−15.689.6
ethcathinone0.10.30.9911−20.916.614.2−4.719.919.214.31.611.616.38.54.3−2.298.8
ethylone0.10.30.9997−12.315.014.5−9.014.619.73.78.618.210.97.23.221.589.5
etizolam0.10.50.992815.0−16.412.58.213.517.214.08.916.617.16.29.8−6.893.8
eutylone0.510.999721.6−15.6−11.0−4.81.714.112.59.719.120.49.19.96.483.5
fentanyl0.050.10.997714.1−20.2−2.6−1.418.519.26.36.819.410.711.29.0−1.190.3
flualprazolam0.20.50.9905−9.918.0−9.90.613.811.811.99.918.318.55.87.2−13.080.2
flunitrazepam0.10.50.998811.3−11.111.07.414.712.514.59.920.111.97.79.221.477.7
fluoxetine0.510.9918−20.819.813.6−9.419.012.22.99.016.816.011.59.74.080.5
flurazepam0.010.050.9954−18.2−12.215.19.517.113.714.79.715.713.87.310.4−7.576.0
furanyl-fentanyl0.10.50.9979−20.818.514.30.319.819.02.410.015.911.214.89.410.676.0
furanyl-norfentanyl0.010.20.995512.6−18.0−12.1−6.318.019.014.19.811.913.49.69.71.386.6
isobutyryl-fentanyl0.10.50.991214.1−17.213.1−8.43.815.715.06.915.519.812.57.24.287.2
isotonitazene0.050.20.998520.6−17.314.9−3.94.93.75.31.512.318.09.17.710.690.6
JWH-0070.510.9991−13.021.4−11.9−4.95.26.714.62.713.715.510.17.627.483.7
JWH-0160.30.10.9999−15.013.3−11.80.719.419.91.76.116.313.215.22.0−4.880.1
JWH-0180.30.10.999714.620.214.23.03.819.414.110.014.113.910.84.71.178.1
JWH-0190.10.50.991312.719.816.4−1.414.91.212.610.012.319.611.910.7−19.876.4
JWH-0730.10.50.9944−16.515.914.5−0.61.73.614.810.016.512.212.72.4−14.694.7
JWH-0810.10.50.9987−19.5−18.212.6−0.919.17.23.19.711.414.112.07.121.493.0
JWH-0980.10.50.9951−13.017.0−7.94.64.719.49.12.910.718.39.19.14.780.0
JWH-1220.10.50.9971−20.619.8−10.4−7.08.211.28.49.112.919.310.35.615.498.9
JWH-1470.020.050.9961−11.7−12.0−4.6−1.919.919.614.79.710.319.712.510.413.284.2
JWH-2000.10.50.9945−19.912.9−14.69.119.619.514.69.911.418.711.810.4−8.289.6
JWH-2030.10.50.9908−12.718.8−13.9−4.15.114.114.45.420.019.611.710.9−4.198.1
JWH-2100.10.50.998518.3−16.2−3.6−6.019.411.85.010.017.317.811.99.510.795.3
JWH-2500.10.50.996516.815.01.6−4.05.219.119.010.020.918.615.09.6−12.093.1
JWH-2510.10.50.9944−13.012.2-12.48.83.21.414.79.619.219.87.72.37.776.2
JWH-3020.10.50.992611.0−18.82.23.618.03.012.610.014.010.08.89.5−11.084.5
JWH-3070.050.10.9934−13.7−18.9−15.03.66.216.214.38.619.116.68.26.5−1.097.1
JWH-3980.10.50.999121.713.814.5−7.119.92.010.98.012.611.111.39.96.382.9
ketamina0.050.20.992612.6−13.1−13.8−5.63.42.28.79.519.119.76.15.5−1.184.1
levomepromazine0.010.10.9914−11.815.810.0−3.119.49.39.15.717.613.510.95.814.689.1
lorazepam0.10.50.999721.8−11.0−8.9−8.42.219.28.12.512.915.97.32.7−11.089.3
lormetazepam0.050.10.999910.9−18.4−12.04.54.219.17.09.519.414.814.89.6−9.392.2
m-CPP0.10.50.9971−12.3−9.6−1.0−9.019.519.46.010.019.813.27.99.510.181.6
MDA0.10.30.997919.919.0−11.0−1.013.24.014.99.714.916.610.25.620.775.6
MDAI0.10.50.994612.4-18.614.82.919.55.22.44.819.012.111.010.4−14.588.9
MDEA0.030.10.991912.70.6−7.40.913.719.014.810.012.015.614.23.2−16.998.4
MDMA0.10.50.9967−20.1−18.6−6.9−1.119.215.914.110.019.413.46.66.2−6.494.9
MDMB-CHMICA0.20.50.9972−17.815.6−13.59.113.27.614.62.017.815.215.47.5−3.780.5
MDPV0.30.50.993518.519.5−12.9−6.414.512.011.29.711.117.013.69.5−6.086.6
MeOAc-fentanyl0.10.50.994219.8−13.22.25.912.714.14.79.019.018.811.35.5−18.396.7
MeOAc-norfentanyl0.10.50.999819.7−11.5−14.51.01.813.714.19.420.920.09.36.3−12.779.2
mephedrone0.10.50.999615.013.35.65.419.912.714.010.020.717.16.39.314.785.8
methamphetamine0.10.50.9997−18.919.50.9−7.215.519.89.79.812.215.48.18.57.996.4
methcathinone0.10.50.997919.620.013.89.119.016.06.02.019.817.311.35.427.790.5
methedrone0.10.50.9992−14.514.4−14.0−3.519.04.014.64.517.619.112.210.2−10.192.1
methoxetamine0.10.50.996419.6−18.814.30.318.92.61.89.415.811.67.05.4−5.394.6
methylone0.10.50.994115.0-18.49.58.714.019.14.79.518.920.76.25.7-13.789.0
midazolam0.050.10.995519.814.6−10.6−2.811.814.214.110.016.813.07.22.48.579.2
mirtazapine0.10.50.9974−18.9−18.114.9−3.018.919.014.99.512.313.012.56.718.179.4
MMB-22010.10.50.996422.015.3−7.1−1.24.519.99.09.912.219.77.28.1−8.991.4
naphyrone0.10.50.997618.1−10.26.29.614.416.85.19.617.319.814.09.42.879.6
N-ethylpentylone0.30.50.990619.120.9−13.8−1.01.21.812.710.013.616.514.46.3−5.898.5
nordiazepam0.10.30.995214.7−13.1−14.7−4.69.47.01.75.915.416.76.37.321.191.1
norfentanyl0.510.997318.813.50.3−8.11.219.014.79.810.710.012.07.210.378.2
norketamina0.10.50.993912.7−16.4−13.1−2.61.913.215.03.610.519.015.19.916.087.4
ocfentanil0.20.10.999418.214.06.19.719.619.24.59.920.016.25.94.5−6.487.9
oxazepam0.510.9966−20.415.310.19.418.919.013.77.519.211.89.99.33.892.1
oxcarbazepine0.510.9988−11.519.912.89.82.46.25.49.419.714.65.66.818.489.3
paroxetine120.998622.413.812.8−4.119.111.214.19.618.220.77.12.9−0.780.8
pentedrone0.10.30.9934−10.4−18.6−13.67.113.619.73.69.419.619.713.48.8−5.179.9
pentylone0.10.30.996617.2−18.614.0−2.819.04.512.59.810.310.514.82.1−12.785.0
pF-furanyl-fentanyl0.010.20.998119.6−9.7−3.6−9.14.720.01.59.516.011.97.58.12.490.0
pinazepam0.010.050.998318.0−9.1−6.0−7.612.615.71.54.510.210.97.97.620.679.7
pravadoline0.020.10.99460.416.15.59.618.42.01.13.520.720.113.910.6−16.382.6
prazepam0.010.020.9981−10.519.51.09.25.419.014.12.911.317.812.49.98.392.0
promazine0.10.50.9944−20.2−17.0−6.6−0.14.219.814.61.319.716.113.97.421.891.7
quetiapine0.10.50.998718.0-20.46.96.419.04.29.72.019.812.011.210.320.187.2
RCS-40.050.10.999416.811.416.86.08.819.62.49.016.812.111.73.58.580.2
RCS-80.020.050.992219.4−18.35.41.01.210.06.19.111.317.311.22.117.079.0
Ritalinic acid0.210.9965−14.2−11.88.20.514.918.212.16.714.317.48.14.815.076.9
temazepam 0.10.50.997822.0−19.0−11.6−8.919.019.914.09.616.313.19.99.521.385.6
trazodone0.10.50.998618.819.310.99.713.912.49.44.819.619.56.88.01.293.6
triazolam0.030.10.998421.5−15.8−14.01.92.414.214.310.015.511.815.54.77.186.6
UR-1440.20.50.999319.214.713.2−6.06.814.17.99.813.419.712.13.4−8.884.1
WIN 55.212-20.050.10.9985−18.819.6−13.9−9.318.319.22.59.911.911.512.08.7−17.297.9
zolpidem0.050.10.9932−17.0−14.813.9−9.015.219.611.79.918.519.912.35.5−3.291.8
zoplicone0.050.10.999122.0−18.714.8−5.44.519.114.52.612.513.29.57.516.794.4
α-OH-alprazolam0.0510.9964−17.1−12.511.65.619.012.414.19.911.814.68.94.5−3.880.9
α-OH-midazolam0.050.10.9977−18.714.5−12.0−7.29.419.714.89.710.311.29.410.1−7.689.9
α-PHP0.50.10.996711.618.08.19.819.49.812.19.818.514.714.87.110.693.6
Table
Table 3. Concentrations found in real cases of consumption.
Table 3. Concentrations found in real cases of consumption.
CaseForensic CaseworkCompoundConcentration (ng/mL)
#1DUIDalprazolam
α-OH-alprazolam		50.39
3.32
#2DUIDdiazepam
nordiazepam
temazepam
oxazepam		346.07
70.82
35.56
15.77
#3DUIDlorazepam85.30
#4DUIDketamina
nor-ketamina		246.37
177.43
#5DUIDdiazepam
nordiazepam
temazepam
oxazepam		1046.36
638.16
18.68
80.93
#6DUIDdiazepam
nordiazepam
temazepam
oxazepam		28.89
74.53
10.58
<LOQ
#7DUIDalprazolam
α-OH-alprazolam		31.30
<LOQ
#8DUIDketamina
nor-ketamina		305.01
198.77
#9DUIDalprazolam
α-OH-alprazolam		7.27
<LOQ
#10DUIDdiazepam
nordiazepam
temazepam
oxazepam		38.25
30.78
29.11
26.89
#11DUIDdiazepam
nordiazepam
temazepam
oxazepam		1830.17
44.00
19.02
<LOQ
#12DUIDlorazepam178.12
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Vaiano, F.; Bertol, E.; Mineo, M.; Pietrosemoli, L.; Rubicondo, J.; Supuran, C.T.; Carta, F. Development of a New LC-MS/MS Screening Method for Detection of 120 NPS and 43 Drugs in Blood. Separations 2021, 8, 221. https://0-doi-org.brum.beds.ac.uk/10.3390/separations8110221

AMA Style

Vaiano F, Bertol E, Mineo M, Pietrosemoli L, Rubicondo J, Supuran CT, Carta F. Development of a New LC-MS/MS Screening Method for Detection of 120 NPS and 43 Drugs in Blood. Separations. 2021; 8(11):221. https://0-doi-org.brum.beds.ac.uk/10.3390/separations8110221

Chicago/Turabian Style

Vaiano, Fabio, Elisabetta Bertol, Maria Mineo, Laura Pietrosemoli, Jolanda Rubicondo, Claudiu T. Supuran, and Fabrizio Carta. 2021. "Development of a New LC-MS/MS Screening Method for Detection of 120 NPS and 43 Drugs in Blood" Separations 8, no. 11: 221. https://0-doi-org.brum.beds.ac.uk/10.3390/separations8110221

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