Research Article
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Detection and quantification of 56 new psychoactive substances in whole blood and urine by LC–MS/MS
Background: New psychoactive substances (NPS) have become increasingly prevalent and are sold in internet shops as ‘bath salts’ or ‘research chemicals’ and comprehensive bioanalytical methods are needed for their detection. Methodology: We developed and validated a method using LC and MS/MS to quantify 56 NPS in blood and urine, including amphetamine derivatives, 2C compounds, aminoindanes, cathinones, piperazines, tryptamines, dissociatives and others. Instrumentation included a Synergi Polar-RP column (Phenomenex) and a 3200 QTrap mass spectrometer (AB Sciex). Run time was 20 min. Conclusion: A novel method is presented for the unambiguous identification and quantification of 56 NPS in blood and urine samples in clinical and forensic cases, e.g., intoxications or driving under the influence of drugs.
From about 2008 onward, new psychoactive substances (NPS) have posed a persistent and constantly growing and evolving international problem [1,2] . NPS can be defined as substances of abuse, ‘that are not controlled by the 1961 Single Convention on Narcotic Drugs or the 1971 Convention on Psychotropic Substances, but which may pose a public health threat’ [2] . This rather broad definition encompasses several different substance classes such as phenethylamines, tryptamines, piperazines, cathinones, synthetic cannabinoid receptor agonists and others [1] . Stimulants, hallucinogens and dissociatives have initially been marketed as ‘bath salts’, but have since been sold under a plethora of different descriptions such as ‘plant food’, ‘wheel shine’ or simply ‘research chemical’. These types of substances are being seized with increasing frequency and have been shown to pose serious health risks [3–5] . Amphetamine derivatives and cathinones can lead to serotonin syndrome [6–9] that can result in coma or death [10–12] . Excited delirium, psychosis, varying effects on memories, cerebral oedema, cardiomyopathy, self-mutilation and increased suicide risk, particularly by hanging and other mechanical methods,
10.4155/BIO.15.48 © 2015 Future Science Ltd
have been reported for cathinones [13–17] . Intravenous application can lead to necrotizing fasciitis [18] . Compounds of the 2C family have been described to have amphetaminelike stimulating effects and mescaline-like hallucinogenic properties [19] . Lethal leukoencephalopathy has been reported following consumption of a 2C compound [20] . Piperazines generally have a stimulating effect and can lead to hyperthermia, tachycardia, renal failure, hallucinations and psychotic episodes, dissociative symptoms, seizures, coma and death [21–23] . Tryptamines are largely hallucinogenic in nature but can also induce tachycardia, bruxism, anxiety and nausea, and have been implicated in several fatalities [24] . Dissociatives like ketamine are often characterized by visual hallucinations, outof-body experiences, impaired consciousness, hypertension and tachycardia, and carry a significantly increased risk of accidents [25] . The legal status of these substances varies widely within Europe and in other regions of the world. Switzerland and Portugal have very restrictive regulations with extensive scheduling of single compounds in place [26–28] . Moreover, in Switzerland, the United Kingdom and the United States analogue laws that only specify certain backbone structures
Bioanalysis (2015) 7(9), 1119–1136
Lars Ambach1, Ana Hernández Redondo1, Stefan König1, Verena Angerer2, Stefan Schürch3 & Wolfgang Weinmann*,1 Institute of Forensic Medicine, University of Bern, Bern, Switzerland 2 Institute of Forensic Medicine, Medical Center, University of Freiburg, Freiburg, Germany 3 Department of Chemistry & Biochemistry, University of Bern, Bern, Switzerland *Author for correspondence: Tel.: +41 (0)31 631 56 68 Fax: +41 (0)316318580 [email protected] 1
part of
ISSN 1757-6180
1119
Research Article Ambach, Hernández Redondo, König, Angerer, Schürch & Weinmann are applied to NPS [26,29–30] . In Austria, an entirely new law separate from existing narcotics legislation, that also regulates analogues, has been created [31,32] . In order to enforce these laws, sensitive methods for the detection and quantitation of NPS are needed. In this publication we will mainly focus on stimulant and hallucinogenic NPS such as amphetamines, cathinones, piperazines or tryptamines. While synthetic cannabinoid receptor agonists make up a significant part of the NPS phenomenon, readers interested in these compounds are kindly referred to the publications of our EU project partners [33–37] . Several methods have been published for the detection of NPS in urine [38–44] , plasma/serum [45–50] , whole blood [51–53] , hair [54–57] , oral fluid [58] , nails [59] , dried blood spots [60] , as well as multiple matrices [61–65] . However, these methods often focus on only one or two substance classes or a limited number of substances [41–47,51–55,58,62,64] . Moreover, methods covering a larger number of substances are often only suitable for qualitative or semiquantitative screening rather than quantification [40,42,50,60,64] . So far, there seems to be a lack of comprehensive analytical methods for the quantification of several groups of NPS in biological samples. We hereby present a fully validated quantitative method for the detection of a comprehensive spectrum of NPS comprised of amphetamine derivatives, 2C compounds, aminoindanes, cathinones, Key terms New psychoactive substances: Substances of abuse, that are not controlled by the 1961 Single Convention on Narcotic Drugs or the 1971 Convention on Psychotropic Substances, but which may pose a public health threat. They have significantly increased in number and popularity since ca. 2008. They were designed to circumvent existing narcotics legislation and were initially marketed as legal products mimicking the effects of controlled substances. Piperazines: In the context of new psychoactive substances, this term refers to either benzylpiperazines or phenylpiperazines with varying degrees of substitution. In general, they have stimulant effects. Cathinones: β-keto derivatives of amphetamines that have generally a stimulant effect, often similar to the corresponding amphetamine. Dissociatives: Structurally diverse class of compounds capable of inducing states of detachment from both the environment of the subject and their own body. Amphetamines: Group of compounds that share a common alpha-methyl-phenethylamine backbone. Depending on the substitution pattern they can have effects ranging from stimulant to entactogen. 2C compounds: Phenethylamines with methoxy groups in the 2- and 5-positions of the phenyl ring.
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piperazines, tryptamines, dissociatives and others in whole blood and urine specimens. Experimental Standards, reagents & samples
2,5-Dimethoxyamphetamine (2,5-DMA), 2C-B, 2C-E, 2C-H, 2C-I, 2C-T-2, 2C-T-4, 2C-T-7, 3,4,5-trimethoxyamphetamine (3,4,5-TMA), 3,4-dimethoxyamphetamine (3,4-DMA), 4-methylethcathinone (4-MEC), 4-methylthioamphetamine (4-MTA), butylone, cathinone, N,N-dimethyltryptamine (DMT), 4-bromo-2,5-dimethoxyamphetamine (DOB), 2,5-dimethoxy-4-ethylamphetamine (DOET), 2,5-dimethoxy-4-methylamphetamine (DOM), N-ethylamphetamine, flephedrone, meta-chlorophenylpiperazine (mCPP), 3,4-methylenedioxy-N,Ndimethylamphetamine (MDDMA), mephedrone and mephedrone-D3, methcathinone, methedrone, methylone, norephedrine, phencyclidine (PCP), paramethoxyamphetamine (PMA), para-methoxymethamphetamine (PMMA), pyrovalerone and 3-trifluoromethylphenylpiperazine (TFMPP) were purchased from Lipomed (Arlesheim, Switzerland). 3-Fluoromethcathinone (3-FMC), amphetamine-D5, cocaineD3, ephedrine, N-ethylcathinone (ethcathinone), ethylone and ethylone-D5, fenfluramine-D10, ketamine-D4, MDEA-D5, MDMA-D5, 3’,4’-methylenedioxy-αpyrrolidinopropiophenone (MDPPP), 3’,4’-methylenedioxypyrovalerone (MDPV), naphyrone and PCP-D5 were ordered from LGC Standards (Wesel, Germany). Benzylpiperazine (BZP), methylenedioxybenzylpiperazine (MDBP) and para-fluorobenzylpiperazine (p-fluoro-BZP) were obtained from Sigma-Aldrich (Buchs, Switzerland). Ketamine was ordered from Goedecke AG (Freiburg, Germany). Para-methoxyphenylpiperazine (MeOPP) and dimethylphenylpiperazine (DMPP), which was used as internal standard (IS), were obtained from VWR (Dietikon, Switzerland). MDPV-D8 was purchased from Adipogen AG (Liestal, Switzerland). 2C-D was ordered from TRC (Toronto, Canada). 2C-P, 5-methoxy-N,N-dimethyltryptamine (5-MeO-DMT), α-methyltryptamine (AMT), N,N-dipropyltryptamine (DPT), N-methylN-isopropyltryptamine (MiPT) and 2,4,6-trimethoxyamphetamine (TMA-6) were kind gifts from the Landeskriminalamt Hamburg, Germany. 5-Iodo2-aminoindane (5-IAI), N,N-diallyl-5-methoxytryptamine (5-MeO-DALT), desoxypipradrol and 5,6-methylenedioxy-2-aminoindane (MDAI) were ordered as ‘research chemicals’ from different internet providers; identities and purities (>98%) were confirmed by 1H and 13C nuclear magnetic resonance spectroscopy and GC–MS. N,N-Diisopropyltryptamine (DiPT) was ordered from Grace (MD, USA).
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Detection & quantification of 56 NPS in blood & urine by LC–MS/MS
Acetic acid (100%, analytical grade), aqueous ammonia (25%, analytical grade), dichloromethane (analytical grade), ethyl acetate (analytical grade), potassium dihydrogenphosphate (KH2PO4, analytical grade), potassium hydroxide pellets (analytical grade), disodium hydrogenphosphate dihydrate (Na 2HPO4 * 2 H2O), sodium hydroxide pellets and hydrochloric acid (fuming, ca. 37%, analytical grade) were purchased from Dr. Grogg Chemie (Bern, Switzerland). HPLC grade water was produced in-house with a Milli-Q water system from Millipore (Zug, Switzerland). Formic acid (analytical grade, 98%) and methanol (spectrophotometric grade, ≥99%) were purchased from Sigma-Aldrich (Buchs, Switzerland). Ammonium formate (analytical grade) was acquired from VWR (Dietikon, Switzerland). 2-Propanol was purchased from Fisher Scientific (Wohlen, Switzerland). Blank human blood for the method validation was obtained from the blood donation center in Bern, Switzerland, blank human urine was donated by employees of the Institute of Forensic Medicine Bern. For the validation of the method’s selectivity blank blood and urine from six different employees of the Institute of Forensic Medicine Bern was used. Preparation of stock solutions, working solutions & buffers
Commercial reference solutions for analytes and IS were available in concentrations of either 0.1 mg/ml or 1.0 mg/ml. A stock solution containing all analytes was prepared by mixing 100 μl of each 0.1 mg/ml methanolic analyte stock solution and 10 μl of each 1.0 mg/ml methanolic analyte stock solution as well as 40 μl of 0.25% HCl in methanol. The mixture was evaporated under a stream of nitrogen at room temperature and reconstituted in 1.0 ml of methanol. The resulting stock solution had a concentration of 10 μg/ml for each analyte. From this stock solution, working solutions with concentrations of 0.025, 0.1, 0.3, 0.5, 1.0, 3.0 and 5.0 μg/ml in methanol were prepared. For the addition of 10 μl volumes, Gilson Microman M10 pipettes were used. At 10 μl, the manufacturer specifies a systematic error of ±0.15 μl (1.5%) and a random error of ≤0.06 μl (0.6%). However, regular air displacement pipettes with the necessary volume range have satisfactory inaccuracy and imprecision for this purpose according to manufacturer’s specifications. An IS stock solution was prepared by mixing 50 μl of each 0.1 mg/ml stock solution, 5 μl of each 1.0 mg/ml stock solution and 1540 μl of methanol. The resulting solution had a concentration of 2.5 μg/ml for each IS. Phosphate buffer pH 6 was prepared by mixing 60.5 ml of 0.066 M Na 2HPO4 solution with 439.5 ml of 0.066 M KH2PO4 solution. Acetate buffer pH 4 was
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produced by adjusting 0.1 M acetic acid to pH 4 with 1 M KOH. Sample preparation
Whole blood samples were processed by solid-phase extraction. First, 500 μl of blood were mixed with 10 μl of IS solution and 2000 μl of phosphate buffer pH 6. The samples were shaken for 5 min and then centrifuged at about 3000 g. Bond Elut Certify solid-phase extraction cartridges (130 mg, 3 ml, Agilent Technologies, Basel, Switzerland) were conditioned with 1 ml of methanol and 1 ml of phosphate buffer pH 6 before the supernatants of the samples were applied under light vacuum. The samples were subsequently washed with 1 ml of water, 1 ml of acetate buffer pH 4, and 1 ml of methanol, and dried under vacuum for 10 min. The analytes were then eluted with 2 ml of dichloromethane/2propanol/ammonia (40:10:1, v/v). The eluate was mixed with 40 μl of 0.25% HCl in methanol and evaporated under a stream of nitrogen at room temperature. The residue was re-dissolved in 50 μl of 10 mM ammonium formate in 0.1% formic acid (mobile phase A). Urine samples were prepared by liquid-liquid extraction (LLE). First, 250 μl of urine were mixed with 10 μl of IS solution and 500 μl of 0.1 M NaOH. The samples were shaken for 5 min, and then 1 ml of ethyl acetate was added. The samples were shaken for another 10 min and centrifuged for 10 min at 16,000 g. The organic phase was transferred into a vial with 20 μl of 0.25% HCl in methanol and evaporated under a stream of nitrogen at room temperature. The residue was re-dissolved in 50 μl of 10 mM ammonium formate in 0.1% formic acid (mobile phase A). Preparation of calibration & control samples
Calibration and control samples were prepared by mixing 450 μl of blank blood with 50 μl of the corresponding working solution and 225 μl of blank urine and 25 μl of working solution, respectively. Calibration and control samples were then processed in the same way as case samples. Calibration samples were prepared with concentrations of 2.5, 10, 50, 100, 500 and 1000 ng/ml while QC samples had concentrations of 30 and 300 ng/ml. Instrumentation & analytical method
The analytical system consisted of an Agilent 1200 HPLC system (Agilent Technologies) equipped with a HTS PAL autosampler (CTC Analytics, Zwingen, Switzerland) and a Synergi Polar-RP column (100 × 2.0 mm, 2.5 μm, Phenomenex, Aschaffenburg,
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Research Article Ambach, Hernández Redondo, König, Angerer, Schürch & Weinmann Germany) maintained at 50 °C and a 3200 QTrap (AB Sciex, Brugg, Switzerland) with Analyst software version 1.5.1 for mass spectrometric detection. For chromatographic separation, mobile phase A consisted of 0.1% formic acid in a 10 mM aqueous solution of ammonium formate, while mobile phase B was 0.1% formic acid in methanol. Gradient elution was performed as follows: 0–5 min: 1% B–7.5% B; 5–12.5 min: 7.5% B–50% B; 12.5–14.5 min: 50% B–90% B; 14.5–16.5 min: 90% B; 16.5–17 min: 90% B–1% B; 17–20 min: 1% B. The flow rate was 0.4 ml/min. In order to improve the sensitivity especially for compounds with early retention times, postcolumn addition of 2-propanol via a t-piece with a flow of 0.2 ml/min was used. The mass spectrometer was operated with positive electrospray ionization and scheduled multiple reaction monitoring (sMRM). The spray voltage was set to 5000 V and the source temperature was set to 400 °C. In general, two transitions were used for each analyte and one transition for each IS. For analytes that had isobaric precursor and fragment mass-to-charge ratios, three transitions were used. The gas settings were: curtain gas: 30, collision gas: 6, gas 1: 40 and gas 2: 60 arbitrary units. The sMRM detection window was set ±30 s around the expected retention time with a target scan time of 1 s. Mass-to-charge ratios for the quantifiers and qualifiers as well as the corresponding potentials and collision energies are summarized in Table 1. Validation
The method has been validated for selectivity and specificity, LLOQ, linearity, imprecision and accuracy, matrix effects, and extraction efficiency following the guidelines of the German Society of Toxicological and Forensic Chemistry (GTFCh) [66] . The selectivity of the method was investigated by analyzing blank blood and urine without added IS from six different individuals as well as two blank samples with IS added for both whole blood and urine. Specificity was tested by injecting single solutions of each analyte and IS with a concentration of 1000 ng/ml in mobile phase A. Compound identification was based on the presence of corresponding signals for qualifier and quantifier transitions, ion ratios as well as retention time. Ion ratios were assessed by comparing the calculated concentrations for quantifiers and qualifiers. In accordance with EU guidelines [67] a maximum deviation of 30% between calculated concentrations was deemed acceptable. The LLOQs were investigated by spiking blank blood and urine with analyte concentrations of 1.0, 2.5, 5.0 and 10 ng/ml and analyzing at least six samples
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per concentration. The LLOQ was defined as the lowest concentration where the relative standard deviation (RSD) as well as the difference of the mean of the calculated concentration from the nominal concentration (bias) was ≤20%. For the evaluation of the linearity of the method, complete calibration curves for blood and urine were analyzed on six consecutive days. The resulting relative peak areas (analyte peak area/IS peak area) were then analyzed using VALISTAT software version 2.00.1 [68] . The Grubbs test was used to check for outliers. No more than one outlier per concentration and two outliers in total for every compound were accepted. Imprecision and accuracy were calculated by analyzing two QC samples of each level on eight consecutive days as required by the GTFCh guidelines [66] . Imprecision was expressed as % RSD of the calculated concentrations and accuracy as the difference of the mean of the calculated concentrations to the nominal concentrations in %. The matrix effect was examined by analyzing blank blood and urine from six different sources with a constant infusion of a solution containing ten analytes selected by retention time (BZP, ethylone, ketamine, MDPPP, MDPV, methcathinone, methylone, naphyrone, norephedrine, pyrovalerone) at a concentration of 1000 ng/ml in mobile phase A into the eluent (10 μl/min flow) by a T-union before it entered the electrospray ion source (postcolumn infusion) [69] . This method for the semiquantitative assessment of matrix effects was originally published by Bonfiglio et al. [70] and its feasibility has also been demonstrated by other researchers [38,71–72] . The extraction efficiency of the method was calculated by comparing the relative peak areas of the analytes of extraction and control samples at concentrations of 30 and 300 ng/ml. The extraction samples (n = 6) were prepared by adding the analytes to blank samples before the extraction step and the IS after the extraction step. In case of the control samples (n = 6) analytes and IS were added after the extraction step. The rest of the sample preparation was carried out as mentioned before. The process of method development and validation is summarized as a flow chart in Figure 1. Results & discussion Method development
A chromatogram of a whole blood QC sample (c = 30 ng/ml) is shown in Figure 2. All analytes eluted between two and 16 min with all isobaric compounds baseline-separated from their isobaric counterparts with the exception of 3-FMC and flephedrone. These two substances are structurally similar and have an
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Detection & quantification of 56 NPS in blood & urine by LC–MS/MS
Research Article
Table 1. MS parameters: precursor ion (Q1), fragment ions (Q3) (quantifier first, qualifiers second and third), expected retention time, declustering potential, entrance potential and collision energy. Collision cell exit potential was 4 V for all transitions. Substance
m/z Q1
m/z Q3
RT (min)
DP (V)
EP (V)
CEP (V)
CE (eV)
2,5-DMA
196.1
179.2/151.1/121.1
10.9
27
7
21
16/22/37
2C-B
260.1
243.0/228.0/213.0
12.9
30
5
14
15/30/41
2C-D
196.2
179.1/ 164.1/149.1
12.0
36
4
8
16/25/35
2C-E
210.2
193.2/ 178.2/163.2
13.8
34
5
21
16/24/38
2C-H
182.0
165.1/ 150.1/135.1
9.0
30
5
22
15/25/39
2C-I
308.1
291.0/ 276.0/261.0
13.9
30
5
18
18/29/40
2C-P
224.1
207.1/ 192.1/163.1
15.1
32
4
24
17/25/37
2C-T-2
242.1
225.1/ 210.1/195.1
13.8
30
4
22
17/27/31
2C-T-4
256.1
239.2/197.1/182.1
14.6
35
4
23
20/28/36
2C-T-7
256.1
239.2/197.1/167.1
15.0
28
4
27
20/29/40
3,4,5-TMA
226.1
209.2/194.2/181.1
9.7
32
4
19
17/25/26
3,4-DMA
196.1
179.1/151.1/107.0
7.8
23
5
19
14/27/50
3-FMC
182.2
164.2/149.1/103.1
5.3
40
5
19
21/29/39
4-MEC
192.1
174.2/144.1
11.1
41
5
19
20/43
4-MTA
182.1
165.1/137.1/117.1
11.8
33
3
18
18/26/27
5-IAI
260.0
116.0/243.0
12.2
45
5
27
38/23
5-MeO-DALT
271.2
110.1/174.1
13.8
36
3
14
19/24
5-MeO-DMT
219.1
174.2/58.0
9.9
29
5
20
21/34
Amphetamine-D5
141.2
93.1
4.9
33
3
16
26
AMT
175.1
158.1/143.1
8.5
22
4
18
16/38
Butylone
222.1
174.2/204.2/146.2
10.4
35
5
22
26/17/34
BZP
177.2
91.1/65.0
3.2
46
4
10
32/62
Cathinone
150.1
132.1/117.1
3.6
36
5
14
19/33
Cocaine-D3
307.2
185.2
13.4
51
5
20
25
Desoxypipradrol
252.2
91.1/167.2
15.1
52
5
9
47/30
DiPT
245.2
144.1/114.1/117.1
13.9
34
6
18
28/20/53
DMPP
191.2
133.1
12.3
55
5
14
35
DMT
189.1
144.1/58.0
8.8
25
5
13
27/29
DOB
274.1
257.1/229.0
13.7
33
4
27
20/28
DOET
224.2
207.1/192.1/177.1
14.6
35
4
24
18/28/37
DOM
210.2
193.1/165.1/178.1
13.1
32
6
20
18/25/27
DPT
245.2
114.1/144.1/86.1
14.4
32
7
14
22/31/38
Ephedrine
166.1
148.1/133.1/115.1
4.1
34
5
18
19/31/39
Ethcathinone
178.1
160.1/130.1/117.1
7.0
40
5
15
18/44/38
2,5-DMA: 2,5-dimethoxyamphetamine; 2C-B: 4-bromo-2,5-dimethoxyphenethylamine; 2C-D: 2,5-dimethoxy-4-methylphenethylamine; 2C-E: 2,5-dimethoxy-4ethylphenethylamine; 2C-H: 2,5-dimethoxyphenethylamine; 2C-I: 2,5-dimethoxy-4-iodophenethylamine; 2C-P: 2,5-dimethoxy-4-n-propylphenethylamine; 2CT-2: 2,5-dimethoxy-4-ethylthiophenethylamine; 2C-T-4: 2,5-dimethoxy-4-isopropylthiophenethylamine; 2C-T-7: 2,5-dimethoxy-4-n-propylthiophenethylamine; 3,4,5-TMA: 3,4,5-trimethoxyamphetamine; 3,4-DMA: 3,4-dimethoxyamphetamine; 4-MEC: 4-methylethcathinone; 4-MTA: 4-methylthioamphetamine; 5-IAI: 5-iodo-2aminoindane; 5-MeO-DALT: N,N-diallyl-5-methoxytryptamine; 5-MeO-DMT: 5-methoxy-N,N-dimethyltryptamine; AMT: α-methyltryptamine; BZP: Benzylpiperazine; CE: Collision energy; CEP: Cell entrance potential; CXP: Cell exit potential; DiPT: N,N-diisopropyltryptamine; DMT: N,N-dimethyltryptamine; DOB: 4-bromo2,5-dimethoxyamphetamine; DOET: 2,5-dimethoxy-4-ethylamphetamine; DOM: 2,5-dimethoxy-4-methylamphetamine; DP: Declustering potential; DPT: N,Ndipropyltryptamine; EP: Entrance potential; mCPP: meta-chlorophenylpiperazine; MDAI: 5,6-methylenedioxy-2-aminoindane; MDBP: Methylenedioxybenzylpiperazine; MDDMA: 3,4-methylenedioxy-N,N-dimethylamphetamine; MDPPP: 3’-4’-methylenedioxy-α-pyrrolidinopropiophenone; MDPV: Methylenedioxypyrovalerone; MeOPP: para-methoxyphenylpiperazine; MiPT: N-methyl-N-isopropyltryptamine; PCP: Phencyclidine; p-fluoro-BZP: para-fluorobenzylpiperazine; PMA: paramethoxyamphetamine, PMMA: para-methoxymethamphetamine; RT: Retention time; TFMPP: 3-trifluoromethylphenylpiperazine; TMA-6: 2,4,6-trimethoxyamphetamine.
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Table 1. MS parameters: precursor ion (Q1), fragment ions (Q3) (quantifier first, qualifiers second and third), expected retention time, declustering potential, entrance potential and collision energy. Collision cell exit potential was 4 V for all transitions (cont.). Substance
m/z Q1
m/z Q3
RT (min)
DP (V)
EP (V)
CEP (V)
CE (eV)
Ethylamphetamine
164.1
91.0/119.0/65.0
9.5
32
6
17
29/17/61
Ethylone
222.1
174.2/204.2 /146.2
9.4
38
5
23
27/21/37
Ethylone-D5
227.2
179.2
9.4
43
5
24
27
Fenfluramine-D10
242.2
161.1
13.2
48
5
20
33
Flephedrone
182.1
164.2/149.1/123.1
5.6
37
5
18
19/30/30
Ketamine
238.1
125.1/220.2
12.1
45
5
21
40/22
Ketamine-D4
242.2
129.0
12.1
42
4
22
38
mCPP
197.1
154.1/118.1
11.7
53
5
21
29/50
MDAI
178.1
161.1/131.1/103.0
5.3
31
5
19
18/26/41
MDBP
221.2
135.1/77.0
4.3
43
5
14
23/57
MDDMA
208.1
163.1/105.1/135.1
10.5
38
6
22
22/37/32
MDEA-D5
213.2
163.1
10.7
38
5
20
19
MDMA-D5
199.1
165.1
9.1
35
5
19
19
MDPPP
248.2
98.1/177.1
11.0
53
4
19
35/23
MDPV
276.2
135.1/126.1
13.8
55
5
25
37/27
MDPV-D8
284.3
134.2
13.8
57
5
22
38
MeOPP
193.1
119.1/150.1
7.8
51
5
20
35/27
Mephedrone
178.1
145.1/160.1 91.1
9.6
36
4
18
28/18/47
Mephedrone-D3
181.1
163.1
9.6
37
8
19
16
Methcathinone
164.1
146.1/130.1/105.1
4.9
38
5
18
19/43/32
Methedrone
194.1
176.2/161.1
8.9
35
5
21
21/28
Methylone
208.1
160.1/132.1/190.1
7.3
31
5
21
24/37/17
MiPT
217.2
86.1/144.1
12.0
28
7
12
18/25
Naphyrone
282.2
141.1/211.1
15.6
57
5
21
37/23
Norephedrine
152.1
134.1/117.1
2.7
29
3
16
15/25
PCP
244.2
91.1/159.2
15.3
29
3.5
22
51/19
PCP-D5
249.2
164.2
15.2
34
4
22
22
p-Fluoro-BZP
195.1
109.0/83.0
4.4
49
5
20
33/64
PMA
166.1
149.1/121.0/91.1
7.8
26
5
19
14/25/44
PMMA
180.2
149.2/121.0
9.6
30
5
20
19/29
Pyrovalerone
246.2
105.1/175.2
14.5
44
5
24
32/24
TFMPP
231.1
188.1/118.1
12.7
51
5
15
30/57
TMA-6
226.1
209.2/181.1/121.1
13.0
24
4
23
18/28/39
2,5-DMA: 2,5-dimethoxyamphetamine; 2C-B: 4-bromo-2,5-dimethoxyphenethylamine; 2C-D: 2,5-dimethoxy-4-methylphenethylamine; 2C-E: 2,5-dimethoxy-4ethylphenethylamine; 2C-H: 2,5-dimethoxyphenethylamine; 2C-I: 2,5-dimethoxy-4-iodophenethylamine; 2C-P: 2,5-dimethoxy-4-n-propylphenethylamine; 2CT-2: 2,5-dimethoxy-4-ethylthiophenethylamine; 2C-T-4: 2,5-dimethoxy-4-isopropylthiophenethylamine; 2C-T-7: 2,5-dimethoxy-4-n-propylthiophenethylamine; 3,4,5-TMA: 3,4,5-trimethoxyamphetamine; 3,4-DMA: 3,4-dimethoxyamphetamine; 4-MEC: 4-methylethcathinone; 4-MTA: 4-methylthioamphetamine; 5-IAI: 5-iodo-2aminoindane; 5-MeO-DALT: N,N-diallyl-5-methoxytryptamine; 5-MeO-DMT: 5-methoxy-N,N-dimethyltryptamine; AMT: α-methyltryptamine; BZP: Benzylpiperazine; CE: Collision energy; CEP: Cell entrance potential; CXP: Cell exit potential; DiPT: N,N-diisopropyltryptamine; DMT: N,N-dimethyltryptamine; DOB: 4-bromo2,5-dimethoxyamphetamine; DOET: 2,5-dimethoxy-4-ethylamphetamine; DOM: 2,5-dimethoxy-4-methylamphetamine; DP: Declustering potential; DPT: N,Ndipropyltryptamine; EP: Entrance potential; mCPP: meta-chlorophenylpiperazine; MDAI: 5,6-methylenedioxy-2-aminoindane; MDBP: Methylenedioxybenzylpiperazine; MDDMA: 3,4-methylenedioxy-N,N-dimethylamphetamine; MDPPP: 3’-4’-methylenedioxy-α-pyrrolidinopropiophenone; MDPV: Methylenedioxypyrovalerone; MeOPP: para-methoxyphenylpiperazine; MiPT: N-methyl-N-isopropyltryptamine; PCP: Phencyclidine; p-fluoro-BZP: para-fluorobenzylpiperazine; PMA: paramethoxyamphetamine, PMMA: para-methoxymethamphetamine; RT: Retention time; TFMPP: 3-trifluoromethylphenylpiperazine; TMA-6: 2,4,6-trimethoxyamphetamine.
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Bioanalysis (2015) 7(9)
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Detection & quantification of 56 NPS in blood & urine by LC–MS/MS
Research Article
Full Scan Q1 Expected [M + H]+ ± 50 amu
Determination of m/z of [M + H]+
EPI spectra (CE 20, 35, 50 eV + CES 35 ± 15 eV)
Spectra for MS/MS library
Optimisation of MRM parameters for 3–4 fragments
Quantitative (s)MRM method 2–3 MRMs per compound
Addition of new compounds influences previous validation, revalidation necessary
New NPS compound
Sample preparation
Validation All validation criteria fulfilled Quantitative
Not all validation criteria fulfilled Semiquantitative
Figure 1. Method development and validation process. Enhanced product ion spectra, acquired at three different collision energies are collected in a database [73] . EPI: Enhanced product ion spectra; NPS: New psychoactive substances; (s)MRM: (scheduled) Multiple reaction monitoring.
almost identical fragmentation pattern. Flephedrone and 3-FMC are not completely baseline-separated (chromatographic resolution, RS ≈ 1.4), but still can be distinguished by the difference of their retention times. The lack of full baseline-separation allows only for semiquantitative detection of these two compounds. A target scan time of 1 s was chosen to ensure a minimum dwell time of at least 15 ms for each transition for optimal sensitivity. At the lowest calibrator, peak width is at least ∼9 s. Therefore, a cycle time of 1 s is sufficient to provide enough data points for proper peak integration. With newer, more sensitive instruments a shorter cycle time might be chosen. Validation
Testing for selectivity and specificity by analyzing blank samples from different sources and solutions of single analytes, respectively, revealed no significant interference from matrix components or cross-talk between different analytes. Ion ratios have been
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summarized in Supplementary Table 1. LLOQs were generally between 1.0 and 10 ng/ml for both matrices. Values for every analyte are listed in Table 2. Calibration curves were linear up to 1000 ng/ml for all analytes (weighting: 1/x, R 2 > 0.99 for all analytes). Regression parameters for all analytes are presented in Supplementary Table 1. Detailed data for imprecision and accuracy are also listed in Table 2. In general, all analytes matched the validation criteria of RSD ≤ 15% and bias ≤ 15% for both QC samples, except for the substances given in Table 3. These compounds can therefore only be analyzed semiquantitatively. Ion suppression was assessed qualitatively by postcolumn infusion. Ten different compounds were chosen for this experiment based on their retention time. Comparison of different analytes shows very similar results on a semiquantitative level. Therefore, analogous results can be expected for the remaining analytes. Exact quantification of matrix effects, following, i.e., the
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Research Article Ambach, Hernández Redondo, König, Angerer, Schürch & Weinmann
B
5.9e5
2.0e5 1.8e5
5.0e5
1.6e5
4.0e5
Intensity (cps)
Intensity (cps)
A
3.0e5 2.0e5 10.87
1.0e5 8.0e4 6.0e4
5
10
15
0.0
20
Time (min)
D 9.0e5
3.0e5
7.0e5
10
15
20
Intensity (cps)
8.0e5
2.5e5 2.0e5 5.16
1.0e5
5.50
6.0e5 5.0e5 4.0e5 3.0e5 13.81
2.0e5
5.0e4 0.0
5
Time (min)
3.8e5 3.5e5
1.5e5
12.83
2.0e4 11.06
C
Intensity (cps)
1.2e5
4.0e4
1.0e5 0.0
1.4e5
1.0e5 5
10
15
20
0.0
Time (min)
5
10
15
20
Time (min)
Figure 2. Extracted ion chromatograms of a whole blood QC sample, c = 30 ng/ml. (A) amphetamines; (B) phenethylamines and aminoindanes; (C) cathinones; (D) piperazines, tryptamines, dissociatives and others.
method of Matuszewski [74] , can be somewhat misleading in the eyes of the authors, as matrix effects vary from sample to sample and the validation process cannot account for all interindividual variances in matrix composition. The only way to really compensate for matrix effects would be the quantification by standard addition (repeated analysis of sample after spiking with different concentrations of the analyte of interest and subsequent back calculation). For whole blood samples (Figure 3A) some periodic ion suppression was seen between 10 and 15 min, presumably by lipophilic matrix compounds. However, there seems to be little to no correlation with the retention times of the compounds listed in Table 3, so this ion suppression appears not to have a significant effect on reproducibility. For urine (Figure 3B), there is no significant matrix effect compared with a solvent blank (Figure 3C). Extraction efficiencies were between 60 and 86% in whole blood and between 57 and 97% in urine. Detailed data is presented in Table 2.
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Bioanalysis (2015) 7(9)
As far as we are aware, most published methods for the detection of NPS focus on single substance groups such as cathinones [42–43,45,51,53,64] , tryptamines [65] , piperazines [41,46] or 2C/DOX substances [44] . Methods covering several substance classes include fewer substances than the method presented here [47,52,58,62] or are intended for qualitative screening purposes rather than quantification [40,42,50,60,64] . More comprehensive methods have been developed by Al-Saffar et al. (27 compounds, urine) [38] , Paul et al. (35 compounds, urine) [39] and Swortwood et al. (32 substances, serum) [49] . Therefore, the presented method for the sensitive detection and quantification of 56 NPS across eight different substance groups in both urine and whole blood samples is a meaningful addition to the collection of tools currently available to forensic and clinical toxicologists. Metabolites & urinalysis
When analyzing urine samples, possible metabolization of the parent analytes should generally be considered.
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Detection & quantification of 56 NPS in blood & urine by LC–MS/MS
Research Article
% Extraction efficiency
% Bias†
Table 2. Validation results. Substance
LLOQ (ng/ml) Blood Urine
Blood
Urine
% RSD Blood
Urine
Blood
Urine
QC 1
QC 2
QC 1
QC 2
QC 1
QC 2
QC 1
QC 2
2,5-DMA
5.00
2.50
79
89
11
9
11
8
7
-13
13
-14
2C-B
2.50
2.50
80
89
11
13
14
13
15
6
6
-6
2C-D
5.00
2.50
86
87
8
8
11
9
-1
-9
9
-12
2C-E
2.50
2.50
85
89
13
14
17
17
27
0
36
-1
2C-H
2.50
2.50
77
79
10
15
12
13
18
3
4
-4
2C-I
2.50
2.50
82
90
8
13
13
14
22
-4
33
-3
2C-P
10.0
2.50
78
91
8
8
10
9
12
-12
12
-17
2C-T-2
2.50
10.0
73
92
12
10
12
15
37
-14
7
-22
2C-T-4
2.50
1.00
72
92
11
11
11
9
0
-2
-14
-12
2C-T-7
2.50
5.00
70
92
14
12
16
13
24
4
-4
-12
3,4,5-TMA
5.00
2.50
82
67
9
15
13
11
6
4
3
-7
3,4-DMA
1.00
2.50
79
61
12
14
12
15
-7
-12
4
-6
4-MEC
5.00
1.00
79
83
10
12
12
12
2
2
-8
-11
4-MTA
2.50
5.00
67
96
14
13
18
22
11
-4
-3
0
5-IAI
5.00
2.50
76
97
14
14
16
13
14
-1
8
-6
5-MeO-DALT
2.50
1.00
66
90
14
11
12
11
2
-3
-21
-16
5-MeO-DMT
2.50
1.00
64
94
11
8
8
6
-13
-14
-6
-13
AMT
2.50
2.50
65
88
12
9
7
9
-14
-16
-15
-12
Butylone
5.00
2.50
78
88
9
11
13
8
6
-3
12
-6
BZP
2.50
2.50
80
77
10
9
9
9
-7
-9
-9
-12
Cathinone
2.50
2.50
79
58
5
12
11
9
1
1
-9
-10
Desoxypipradrol
2.50
1.00
77
91
8
7
9
8
-4
-9
-7
-16
DiPT
2.50
1.00
64
92
13
12
9
16
-11
-13
-10
-16
DMT
5.00
2.50
69
95
12
10
11
11
12
3
-1
-7
DOB
5.00
2.50
81
94
13
11
11
9
-1
-11
0
-11
DOET
2.50
1.00
79
89
8
10
12
12
30
-5
-5
-12
DOM
2.50
2.50
79
94
5
8
9
8
8
-8
9
-11
DPT
2.50
1.00
63
92
10
7
6
8
-6
-12
-10
-15
Ephedrine
2.50
2.50
79
70
10
11
9
12
-5
-11
-7
-12
Ethcathinone
2.50
2.50
75
72
10
11
9
12
-8
-12
-10
-8
%bias: relative difference of mean from nominal value in %. 3-FMC and flephedrone were not baseline-separated and determined only semiquantitatively. Therefore, no validation data is available for these substances. 2,5-DMA: 2,5-dimethoxyamphetamine; 2C-B: 4-bromo-2,5-dimethoxyphenethylamine; 2C-D: 2,5-dimethoxy-4-methylphenethylamine; 2C-E: 2,5-dimethoxy4-ethylphenethylamine; 2C-H: 2,5-dimethoxyphenethylamine; 2C-I: 2,5-dimethoxy-4-iodophenethylamine; 2C-P: 2,5-dimethoxy-4-n-propylphenethylamine; 2C-T-2: 2,5-dimethoxy-4-ethylthiophenethylamine; 2C-T-4: 2,5-dimethoxy-4-isopropylthiophenethylamine; 2C-T-7: 2,5-dimethoxy-4-n-propylthiophenethylamine; 3,4,5-TMA: 3,4,5-trimethoxyamphetamine; 3,4-DMA: 3,4-dimethoxyamphetamine; 4-MEC: 4-methylethcathinone; 4-MTA: 4-methylthioamphetamine; 5-IAI: 5-iodo-2-aminoindane; 5-MeO-DALT: N,N-diallyl-5-methoxytryptamine; 5-MeO-DMT: 5-methoxy-N,N-dimethyltryptamine; AMT: α-methyltryptamine; BZP: benzylpiperazine; DiPT: N,N-diisopropyltryptamine; DMT: N,N-dimethyltryptamine; DOB: 4-bromo-2,5-dimethoxyamphetamine; DOET: 2,5-dimethoxy-4ethylamphetamine; DOM: 2,5-dimethoxy-4-methylamphetamine; DPT: N,N-dipropyltryptamine; mCPP: meta-chlorophenylpiperazine; MDAI: 5,6-methylenedioxy2-aminoindane; MDBP: methylenedioxybenzylpiperazine; MDDMA: 3,4-methylenedioxy-N,N-dimethylamphetamine; MDPPP: 3’-4’-methylenedioxyα-pyrrolidinopropiophenone; MDPV: methylenedioxypyrovalerone; MeOPP: para-methoxyphenylpiperazine; MiPT: N-methyl-N-isopropyltryptamine; PCP: phencyclidine; p-fluoro-BZP: para-fluorobenzylpiperazine; PMA: para-methoxyamphetamine, PMMA: para-methoxymethamphetamine; TFMPP: 3-trifluoromethylphenylpiperazine; TMA-6: 2,4,6-trimethoxyamphetamine. †
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Research Article Ambach, Hernández Redondo, König, Angerer, Schürch & Weinmann
Table 2. Validation results (cont.). Substance
LLOQ (ng/ml)
% Extraction efficiency
% RSD Blood
% Bias†
Blood Urine
Blood
Urine
QC 1
QC 2
QC 1
Urine QC 2
QC 1
Blood QC 2
QC 1
Urine QC 2
Ethylamphetamine 2.50
2.50
82
89
11
13
14
11
9
5
11
1
Ethylone
2.50
1.00
79
91
7
8
8
7
1
-11
-2
-12
Ketamine
2.50
1.00
78
95
12
7
8
6
-7
-15
-5
-14
mCPP
2.50
2.50
80
95
11
13
11
10
14
3
9
-4
MDAI
1.00
2.50
77
91
10
11
8
10
-5
-20
-5
-10
MDBP
2.50
2.50
76
86
10
10
10
11
-13
-12
-5
-9
MDDMA
2.50
2.50
75
92
9
12
9
9
1
-9
0
-8
MDPPP
2.50
1.00
80
93
12
10
9
7
2
-2
-5
-14
MDPV
5.00
2.50
82
96
7
10
8
7
9
-5
4
-11
MeOPP
2.50
1.00
60
80
12
11
12
6
4
-3
-10
-17
Mephedrone
2.50
2.50
81
81
8
13
13
10
5
4
6
-2
Methcathinone
2.50
1.00
81
73
8
11
12
11
13
7
-7
-7
Methedrone
2.50
2.50
80
82
7
11
14
11
0
-1
6
-4
Methylone
2.50
1.00
78
84
8
11
11
11
2
3
-8
-8
MiPT
2.50
2.50
65
94
9
8
9
8
-8
-14
-4
-13
Naphyrone
2.50
1.00
77
87
7
9
9
10
4
-6
-12
-14
Norephedrine
2.50
5.00
77
57
9
14
15
13
17
-4
-2
-5
PCP
2.50
2.50
81
90
6
10
8
7
12
-1
1
-7
p-Fluoro-BZP
2.50
2.50
76
86
13
8
7
12
-2
-15
-5
-15
PMA
2.50
1.00
79
88
8
10
11
11
2
-11
-9
-14
PMMA
2.50
2.50
78
90
7
8
7
8
11
-17
6
-13
Pyrovalerone
2.50
1.00
76
90
6
10
12
10
-1
-2
-7
-11
TFMPP
2.50
1.00
73
96
10
10
10
9
-15
-6
-11
-8
TMA-6
5.00
2.50
78
78
8
10
9
9
-1
-3
2
-9
%bias: relative difference of mean from nominal value in %. 3-FMC and flephedrone were not baseline-separated and determined only semiquantitatively. Therefore, no validation data is available for these substances. 2,5-DMA: 2,5-dimethoxyamphetamine; 2C-B: 4-bromo-2,5-dimethoxyphenethylamine; 2C-D: 2,5-dimethoxy-4-methylphenethylamine; 2C-E: 2,5-dimethoxy4-ethylphenethylamine; 2C-H: 2,5-dimethoxyphenethylamine; 2C-I: 2,5-dimethoxy-4-iodophenethylamine; 2C-P: 2,5-dimethoxy-4-n-propylphenethylamine; 2C-T-2: 2,5-dimethoxy-4-ethylthiophenethylamine; 2C-T-4: 2,5-dimethoxy-4-isopropylthiophenethylamine; 2C-T-7: 2,5-dimethoxy-4-n-propylthiophenethylamine; 3,4,5-TMA: 3,4,5-trimethoxyamphetamine; 3,4-DMA: 3,4-dimethoxyamphetamine; 4-MEC: 4-methylethcathinone; 4-MTA: 4-methylthioamphetamine; 5-IAI: 5-iodo-2-aminoindane; 5-MeO-DALT: N,N-diallyl-5-methoxytryptamine; 5-MeO-DMT: 5-methoxy-N,N-dimethyltryptamine; AMT: α-methyltryptamine; BZP: benzylpiperazine; DiPT: N,N-diisopropyltryptamine; DMT: N,N-dimethyltryptamine; DOB: 4-bromo-2,5-dimethoxyamphetamine; DOET: 2,5-dimethoxy-4ethylamphetamine; DOM: 2,5-dimethoxy-4-methylamphetamine; DPT: N,N-dipropyltryptamine; mCPP: meta-chlorophenylpiperazine; MDAI: 5,6-methylenedioxy2-aminoindane; MDBP: methylenedioxybenzylpiperazine; MDDMA: 3,4-methylenedioxy-N,N-dimethylamphetamine; MDPPP: 3’-4’-methylenedioxyα-pyrrolidinopropiophenone; MDPV: methylenedioxypyrovalerone; MeOPP: para-methoxyphenylpiperazine; MiPT: N-methyl-N-isopropyltryptamine; PCP: phencyclidine; p-fluoro-BZP: para-fluorobenzylpiperazine; PMA: para-methoxyamphetamine, PMMA: para-methoxymethamphetamine; TFMPP: 3-trifluoromethylphenylpiperazine; TMA-6: 2,4,6-trimethoxyamphetamine. †
While synthetic cannabinoids show extensive metabolism to the point where most of the time only metabolites are detectable in urine samples, cathinones are metabolized to a far lesser degree [75] . In the case of cathinones and piperazines, parent compounds are generally abundant in urine, for piperazines even in higher concentrations than their respective metabolites [76] . A reported intoxication with 5-MeO-DiPT, where significant concentrations of the parent
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Bioanalysis (2015) 7(9)
compound (229 ng/ml) were present in urine [62] , indicates that this is also true for tryptamines. Therefore, focusing on parent compounds should be sufficient for the detection of stimulant NPS in urine, especially as there are still little pharmacokinetic data available for such compounds and even less reference standards for the quantification of their metabolites. It should be self-evident, however, that the inclusion of metabolites, such as the corresponding ephedrines for
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Detection & quantification of 56 NPS in blood & urine by LC–MS/MS
Research Article
Table 3. Substances that can only be determined semiquantitatively. Semiquantitatively in urine
Semiquantitatively in whole blood Semiquantitatively in both matrices
2C-P
2C-H
2C-E
4-MTA
AMT
2C-I
5-IAI
DOET
2C-T-2
5-MeO-DALT
MDAI
2C-T-7
Desoxypipradrol
Norephedrine
3-FMC
DiPT
PMMA
Flephedrone
MeOPP
cathinones, may further improve the detection of NPS consumption. Unfortunately, at the time of method development, such substances were not available to us as reference standards. Another issue that should be taken into account is the fact that some of the parent compounds themselves may be metabolites of other substances. Ephedrine and Norephedrine can be formed by either metabolic reduction of methcathinone and cathinone, respectively, or oxidative metabolization of methamphetamine and amphetamine, respectively. Piperazine derivatives may be formed from several pharmaceutical drugs: mCPP is a known metabolite of trazodone, MDBP may be formed from piribedil or fipexide while BZP is a metabolite of befuraline and piberaline. 2C compounds may be formed from their NBOMe derivatives by N-dealkylation. Parent compounds and metabolites may be detectable for a longer timeframe in urine compared with blood samples. Analysis of blood samples
Although concentrations of active compounds may be lower in blood than in urine, in the forensic field impairment due to drug consumption needs to be assessed. This is usually underpinned by concentrations of the active compound in blood. In postmortem cases with suspected intoxications, analysis of blood samples is important to determine the cause of death; urine – if available – is used for screening only. Depending on the jurisdiction, urine may only be used for preliminary testing by the police while blood is sent to a forensic laboratory for confirmatory analysis. Application to authentic samples
The developed method has also been applied to authentic samples and some positive results are presented in Table 4. In these cases the most common substance is cathinone. In almost all cases (except case 1) parent compounds are present in both whole blood and urine. Norephedrine is a metabolite of cathinone generated by a reduction of the keto group to a hydroxyl group and
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is also present in all cases involving cathinone (cases 2–5, 8). In an analogous fashion ephedrine is a metabolite of methcathinone (case 8). Norephedrine can also appear as an oxidative metabolite of amphetamine (cases 1 and 7). In cases 1 and 5 ketamine was detected. The first case involved a traffic control following suspicious driving behavior indicating a recreational use of ketamine. The second case on the other hand involved a heavy traffic accident where administration of ketamine by paramedics or emergency physicians is a plausible scenario although no information in this regard was supplied by police. Continued method development
One of the distinctive characteristics of the NPS market is its ever-changing nature. Compounds that are included into the narcotics legislation often decline/ disappear from the markets (with the exception of a few compounds) and new substances are introduced as their replacements [77] . Therefore, method adaption and expansion is a necessity. Being MRM-based, the presented method can easily be modified by editing the list of MRM transitions. This, however, poses the question of revalidation. L Huber explicitly suggests method revalidation when ‘[n]ew compounds are analyzed that are not within the method’s original scope’ [78] and the SWGTOX guidelines [79] recommend revalidating the parameters that are affected by the change to the method. In the case of including new analytes this would obviously entail revalidating selectivity and specificity for all compounds. Moreover, if a regular MRM approach is used, additional compounds will increase the cycle time which in turn might result in less data points per peak and therefore deteriorating peak shape. Thus, reproducibility might be negatively affected and RSD and bias should be re-evaluated for all compounds. In case of an sMRM method with a set cycle time, the addition of new analytes will lower the dwell times for compounds that also elute in the new compounds’ retention time window. This can potentially affect sensitivity.
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1129
Research Article Ambach, Hernández Redondo, König, Angerer, Schürch & Weinmann Therefore, the LODs and/or LLOQs for all compounds with retention time windows overlapping with the new analytes would need to be revalidated. Since these criteria do not have to be checked for all
analytes, the use of sMRM might be more efficient for method expansion. Naturally, for the new substances all validation criteria need to be checked. Expanding and subsequently revalidating a multianalyte method
A 1.4e5
1.2e5
Intensity (cps)
1.0e5
8.0e4
6.0e4
4.0e4
2.0e4 0 .3 0 0.63
2.19
2.47
3.47
0.0
4.40 5.07
5.85
6.59 6.83 7.71 7 .9 4 9.22
9.73
5
10.23
11.86
12.31
14.00
10
14.52
15.21
17.71 17.92
18.35
15
20
Time (min) B 1.4e5
1.2e5
Intensity (cps)
1.0e5
8.0e4
6.0e4
4.0e4
2.0e4 0.62
0.99
2.58
3.39
5.49
4.184.89
6.65 7.30
7.62
8.54 9.20
10.12
11.49
11.70
12.89
13.93
14.32
0.0 5
10
15
15.21
15.94
17.95
18.31
19.70
20
Time (min) Figure 3. Test for matrix effects. Ion suppression by postcolumn infusion in (A) whole blood samples, (B) urine samples and (C) solvent blank samples.
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Bioanalysis (2015) 7(9)
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Detection & quantification of 56 NPS in blood & urine by LC–MS/MS
Research Article
C 2.0e5 1.8e5 1.6e5
Intensity (cps)
1.4e5 1.2e5 1.0e5 8.0e4 6.0e4 4.0e4 2.0e4 0.0 5
10
15
20
Time (min) Figure 3. Test for matrix effects (cont.). Ion suppression by postcolumn infusion in (A) whole blood samples, (B) urine samples and (C) solvent blank samples (cont.).
might consequently prove to be a rather time consuming effort depending on the number of analytes and whether MRM or sMRM is used. The minimum of revalidation should include the following: Linearity of concentration-response
relationship should be investigated in the expected concentration range of the new analytes. Specificity and selectivity should be confirmed by analyzing different blank matrix samples and samples spiked with the previously included analytes (e.g., old calibrators
Table 4. New psychoactive substances found in matching urine and whole blood samples. Sample (n)
NPS in urine
NPS in whole blood
1
Ketamine, norephedrine
Amphetamine (
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Detection and quantification of 56 new psychoactive substances in whole blood and urine by LC–MS/MS
Background: New psychoactive substances (NPS) have become increasingly prevalent and are sold in internet shops as ‘bath salts’ or ‘research chemicals’ and comprehensive bioanalytical methods are needed for their detection. Methodology: We developed and validated a method using LC and MS/MS to quantify 56 NPS in blood and urine, including amphetamine derivatives, 2C compounds, aminoindanes, cathinones, piperazines, tryptamines, dissociatives and others. Instrumentation included a Synergi Polar-RP column (Phenomenex) and a 3200 QTrap mass spectrometer (AB Sciex). Run time was 20 min. Conclusion: A novel method is presented for the unambiguous identification and quantification of 56 NPS in blood and urine samples in clinical and forensic cases, e.g., intoxications or driving under the influence of drugs.
From about 2008 onward, new psychoactive substances (NPS) have posed a persistent and constantly growing and evolving international problem [1,2] . NPS can be defined as substances of abuse, ‘that are not controlled by the 1961 Single Convention on Narcotic Drugs or the 1971 Convention on Psychotropic Substances, but which may pose a public health threat’ [2] . This rather broad definition encompasses several different substance classes such as phenethylamines, tryptamines, piperazines, cathinones, synthetic cannabinoid receptor agonists and others [1] . Stimulants, hallucinogens and dissociatives have initially been marketed as ‘bath salts’, but have since been sold under a plethora of different descriptions such as ‘plant food’, ‘wheel shine’ or simply ‘research chemical’. These types of substances are being seized with increasing frequency and have been shown to pose serious health risks [3–5] . Amphetamine derivatives and cathinones can lead to serotonin syndrome [6–9] that can result in coma or death [10–12] . Excited delirium, psychosis, varying effects on memories, cerebral oedema, cardiomyopathy, self-mutilation and increased suicide risk, particularly by hanging and other mechanical methods,
10.4155/BIO.15.48 © 2015 Future Science Ltd
have been reported for cathinones [13–17] . Intravenous application can lead to necrotizing fasciitis [18] . Compounds of the 2C family have been described to have amphetaminelike stimulating effects and mescaline-like hallucinogenic properties [19] . Lethal leukoencephalopathy has been reported following consumption of a 2C compound [20] . Piperazines generally have a stimulating effect and can lead to hyperthermia, tachycardia, renal failure, hallucinations and psychotic episodes, dissociative symptoms, seizures, coma and death [21–23] . Tryptamines are largely hallucinogenic in nature but can also induce tachycardia, bruxism, anxiety and nausea, and have been implicated in several fatalities [24] . Dissociatives like ketamine are often characterized by visual hallucinations, outof-body experiences, impaired consciousness, hypertension and tachycardia, and carry a significantly increased risk of accidents [25] . The legal status of these substances varies widely within Europe and in other regions of the world. Switzerland and Portugal have very restrictive regulations with extensive scheduling of single compounds in place [26–28] . Moreover, in Switzerland, the United Kingdom and the United States analogue laws that only specify certain backbone structures
Bioanalysis (2015) 7(9), 1119–1136
Lars Ambach1, Ana Hernández Redondo1, Stefan König1, Verena Angerer2, Stefan Schürch3 & Wolfgang Weinmann*,1 Institute of Forensic Medicine, University of Bern, Bern, Switzerland 2 Institute of Forensic Medicine, Medical Center, University of Freiburg, Freiburg, Germany 3 Department of Chemistry & Biochemistry, University of Bern, Bern, Switzerland *Author for correspondence: Tel.: +41 (0)31 631 56 68 Fax: +41 (0)316318580 [email protected] 1
part of
ISSN 1757-6180
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Research Article Ambach, Hernández Redondo, König, Angerer, Schürch & Weinmann are applied to NPS [26,29–30] . In Austria, an entirely new law separate from existing narcotics legislation, that also regulates analogues, has been created [31,32] . In order to enforce these laws, sensitive methods for the detection and quantitation of NPS are needed. In this publication we will mainly focus on stimulant and hallucinogenic NPS such as amphetamines, cathinones, piperazines or tryptamines. While synthetic cannabinoid receptor agonists make up a significant part of the NPS phenomenon, readers interested in these compounds are kindly referred to the publications of our EU project partners [33–37] . Several methods have been published for the detection of NPS in urine [38–44] , plasma/serum [45–50] , whole blood [51–53] , hair [54–57] , oral fluid [58] , nails [59] , dried blood spots [60] , as well as multiple matrices [61–65] . However, these methods often focus on only one or two substance classes or a limited number of substances [41–47,51–55,58,62,64] . Moreover, methods covering a larger number of substances are often only suitable for qualitative or semiquantitative screening rather than quantification [40,42,50,60,64] . So far, there seems to be a lack of comprehensive analytical methods for the quantification of several groups of NPS in biological samples. We hereby present a fully validated quantitative method for the detection of a comprehensive spectrum of NPS comprised of amphetamine derivatives, 2C compounds, aminoindanes, cathinones, Key terms New psychoactive substances: Substances of abuse, that are not controlled by the 1961 Single Convention on Narcotic Drugs or the 1971 Convention on Psychotropic Substances, but which may pose a public health threat. They have significantly increased in number and popularity since ca. 2008. They were designed to circumvent existing narcotics legislation and were initially marketed as legal products mimicking the effects of controlled substances. Piperazines: In the context of new psychoactive substances, this term refers to either benzylpiperazines or phenylpiperazines with varying degrees of substitution. In general, they have stimulant effects. Cathinones: β-keto derivatives of amphetamines that have generally a stimulant effect, often similar to the corresponding amphetamine. Dissociatives: Structurally diverse class of compounds capable of inducing states of detachment from both the environment of the subject and their own body. Amphetamines: Group of compounds that share a common alpha-methyl-phenethylamine backbone. Depending on the substitution pattern they can have effects ranging from stimulant to entactogen. 2C compounds: Phenethylamines with methoxy groups in the 2- and 5-positions of the phenyl ring.
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piperazines, tryptamines, dissociatives and others in whole blood and urine specimens. Experimental Standards, reagents & samples
2,5-Dimethoxyamphetamine (2,5-DMA), 2C-B, 2C-E, 2C-H, 2C-I, 2C-T-2, 2C-T-4, 2C-T-7, 3,4,5-trimethoxyamphetamine (3,4,5-TMA), 3,4-dimethoxyamphetamine (3,4-DMA), 4-methylethcathinone (4-MEC), 4-methylthioamphetamine (4-MTA), butylone, cathinone, N,N-dimethyltryptamine (DMT), 4-bromo-2,5-dimethoxyamphetamine (DOB), 2,5-dimethoxy-4-ethylamphetamine (DOET), 2,5-dimethoxy-4-methylamphetamine (DOM), N-ethylamphetamine, flephedrone, meta-chlorophenylpiperazine (mCPP), 3,4-methylenedioxy-N,Ndimethylamphetamine (MDDMA), mephedrone and mephedrone-D3, methcathinone, methedrone, methylone, norephedrine, phencyclidine (PCP), paramethoxyamphetamine (PMA), para-methoxymethamphetamine (PMMA), pyrovalerone and 3-trifluoromethylphenylpiperazine (TFMPP) were purchased from Lipomed (Arlesheim, Switzerland). 3-Fluoromethcathinone (3-FMC), amphetamine-D5, cocaineD3, ephedrine, N-ethylcathinone (ethcathinone), ethylone and ethylone-D5, fenfluramine-D10, ketamine-D4, MDEA-D5, MDMA-D5, 3’,4’-methylenedioxy-αpyrrolidinopropiophenone (MDPPP), 3’,4’-methylenedioxypyrovalerone (MDPV), naphyrone and PCP-D5 were ordered from LGC Standards (Wesel, Germany). Benzylpiperazine (BZP), methylenedioxybenzylpiperazine (MDBP) and para-fluorobenzylpiperazine (p-fluoro-BZP) were obtained from Sigma-Aldrich (Buchs, Switzerland). Ketamine was ordered from Goedecke AG (Freiburg, Germany). Para-methoxyphenylpiperazine (MeOPP) and dimethylphenylpiperazine (DMPP), which was used as internal standard (IS), were obtained from VWR (Dietikon, Switzerland). MDPV-D8 was purchased from Adipogen AG (Liestal, Switzerland). 2C-D was ordered from TRC (Toronto, Canada). 2C-P, 5-methoxy-N,N-dimethyltryptamine (5-MeO-DMT), α-methyltryptamine (AMT), N,N-dipropyltryptamine (DPT), N-methylN-isopropyltryptamine (MiPT) and 2,4,6-trimethoxyamphetamine (TMA-6) were kind gifts from the Landeskriminalamt Hamburg, Germany. 5-Iodo2-aminoindane (5-IAI), N,N-diallyl-5-methoxytryptamine (5-MeO-DALT), desoxypipradrol and 5,6-methylenedioxy-2-aminoindane (MDAI) were ordered as ‘research chemicals’ from different internet providers; identities and purities (>98%) were confirmed by 1H and 13C nuclear magnetic resonance spectroscopy and GC–MS. N,N-Diisopropyltryptamine (DiPT) was ordered from Grace (MD, USA).
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Detection & quantification of 56 NPS in blood & urine by LC–MS/MS
Acetic acid (100%, analytical grade), aqueous ammonia (25%, analytical grade), dichloromethane (analytical grade), ethyl acetate (analytical grade), potassium dihydrogenphosphate (KH2PO4, analytical grade), potassium hydroxide pellets (analytical grade), disodium hydrogenphosphate dihydrate (Na 2HPO4 * 2 H2O), sodium hydroxide pellets and hydrochloric acid (fuming, ca. 37%, analytical grade) were purchased from Dr. Grogg Chemie (Bern, Switzerland). HPLC grade water was produced in-house with a Milli-Q water system from Millipore (Zug, Switzerland). Formic acid (analytical grade, 98%) and methanol (spectrophotometric grade, ≥99%) were purchased from Sigma-Aldrich (Buchs, Switzerland). Ammonium formate (analytical grade) was acquired from VWR (Dietikon, Switzerland). 2-Propanol was purchased from Fisher Scientific (Wohlen, Switzerland). Blank human blood for the method validation was obtained from the blood donation center in Bern, Switzerland, blank human urine was donated by employees of the Institute of Forensic Medicine Bern. For the validation of the method’s selectivity blank blood and urine from six different employees of the Institute of Forensic Medicine Bern was used. Preparation of stock solutions, working solutions & buffers
Commercial reference solutions for analytes and IS were available in concentrations of either 0.1 mg/ml or 1.0 mg/ml. A stock solution containing all analytes was prepared by mixing 100 μl of each 0.1 mg/ml methanolic analyte stock solution and 10 μl of each 1.0 mg/ml methanolic analyte stock solution as well as 40 μl of 0.25% HCl in methanol. The mixture was evaporated under a stream of nitrogen at room temperature and reconstituted in 1.0 ml of methanol. The resulting stock solution had a concentration of 10 μg/ml for each analyte. From this stock solution, working solutions with concentrations of 0.025, 0.1, 0.3, 0.5, 1.0, 3.0 and 5.0 μg/ml in methanol were prepared. For the addition of 10 μl volumes, Gilson Microman M10 pipettes were used. At 10 μl, the manufacturer specifies a systematic error of ±0.15 μl (1.5%) and a random error of ≤0.06 μl (0.6%). However, regular air displacement pipettes with the necessary volume range have satisfactory inaccuracy and imprecision for this purpose according to manufacturer’s specifications. An IS stock solution was prepared by mixing 50 μl of each 0.1 mg/ml stock solution, 5 μl of each 1.0 mg/ml stock solution and 1540 μl of methanol. The resulting solution had a concentration of 2.5 μg/ml for each IS. Phosphate buffer pH 6 was prepared by mixing 60.5 ml of 0.066 M Na 2HPO4 solution with 439.5 ml of 0.066 M KH2PO4 solution. Acetate buffer pH 4 was
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produced by adjusting 0.1 M acetic acid to pH 4 with 1 M KOH. Sample preparation
Whole blood samples were processed by solid-phase extraction. First, 500 μl of blood were mixed with 10 μl of IS solution and 2000 μl of phosphate buffer pH 6. The samples were shaken for 5 min and then centrifuged at about 3000 g. Bond Elut Certify solid-phase extraction cartridges (130 mg, 3 ml, Agilent Technologies, Basel, Switzerland) were conditioned with 1 ml of methanol and 1 ml of phosphate buffer pH 6 before the supernatants of the samples were applied under light vacuum. The samples were subsequently washed with 1 ml of water, 1 ml of acetate buffer pH 4, and 1 ml of methanol, and dried under vacuum for 10 min. The analytes were then eluted with 2 ml of dichloromethane/2propanol/ammonia (40:10:1, v/v). The eluate was mixed with 40 μl of 0.25% HCl in methanol and evaporated under a stream of nitrogen at room temperature. The residue was re-dissolved in 50 μl of 10 mM ammonium formate in 0.1% formic acid (mobile phase A). Urine samples were prepared by liquid-liquid extraction (LLE). First, 250 μl of urine were mixed with 10 μl of IS solution and 500 μl of 0.1 M NaOH. The samples were shaken for 5 min, and then 1 ml of ethyl acetate was added. The samples were shaken for another 10 min and centrifuged for 10 min at 16,000 g. The organic phase was transferred into a vial with 20 μl of 0.25% HCl in methanol and evaporated under a stream of nitrogen at room temperature. The residue was re-dissolved in 50 μl of 10 mM ammonium formate in 0.1% formic acid (mobile phase A). Preparation of calibration & control samples
Calibration and control samples were prepared by mixing 450 μl of blank blood with 50 μl of the corresponding working solution and 225 μl of blank urine and 25 μl of working solution, respectively. Calibration and control samples were then processed in the same way as case samples. Calibration samples were prepared with concentrations of 2.5, 10, 50, 100, 500 and 1000 ng/ml while QC samples had concentrations of 30 and 300 ng/ml. Instrumentation & analytical method
The analytical system consisted of an Agilent 1200 HPLC system (Agilent Technologies) equipped with a HTS PAL autosampler (CTC Analytics, Zwingen, Switzerland) and a Synergi Polar-RP column (100 × 2.0 mm, 2.5 μm, Phenomenex, Aschaffenburg,
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Research Article Ambach, Hernández Redondo, König, Angerer, Schürch & Weinmann Germany) maintained at 50 °C and a 3200 QTrap (AB Sciex, Brugg, Switzerland) with Analyst software version 1.5.1 for mass spectrometric detection. For chromatographic separation, mobile phase A consisted of 0.1% formic acid in a 10 mM aqueous solution of ammonium formate, while mobile phase B was 0.1% formic acid in methanol. Gradient elution was performed as follows: 0–5 min: 1% B–7.5% B; 5–12.5 min: 7.5% B–50% B; 12.5–14.5 min: 50% B–90% B; 14.5–16.5 min: 90% B; 16.5–17 min: 90% B–1% B; 17–20 min: 1% B. The flow rate was 0.4 ml/min. In order to improve the sensitivity especially for compounds with early retention times, postcolumn addition of 2-propanol via a t-piece with a flow of 0.2 ml/min was used. The mass spectrometer was operated with positive electrospray ionization and scheduled multiple reaction monitoring (sMRM). The spray voltage was set to 5000 V and the source temperature was set to 400 °C. In general, two transitions were used for each analyte and one transition for each IS. For analytes that had isobaric precursor and fragment mass-to-charge ratios, three transitions were used. The gas settings were: curtain gas: 30, collision gas: 6, gas 1: 40 and gas 2: 60 arbitrary units. The sMRM detection window was set ±30 s around the expected retention time with a target scan time of 1 s. Mass-to-charge ratios for the quantifiers and qualifiers as well as the corresponding potentials and collision energies are summarized in Table 1. Validation
The method has been validated for selectivity and specificity, LLOQ, linearity, imprecision and accuracy, matrix effects, and extraction efficiency following the guidelines of the German Society of Toxicological and Forensic Chemistry (GTFCh) [66] . The selectivity of the method was investigated by analyzing blank blood and urine without added IS from six different individuals as well as two blank samples with IS added for both whole blood and urine. Specificity was tested by injecting single solutions of each analyte and IS with a concentration of 1000 ng/ml in mobile phase A. Compound identification was based on the presence of corresponding signals for qualifier and quantifier transitions, ion ratios as well as retention time. Ion ratios were assessed by comparing the calculated concentrations for quantifiers and qualifiers. In accordance with EU guidelines [67] a maximum deviation of 30% between calculated concentrations was deemed acceptable. The LLOQs were investigated by spiking blank blood and urine with analyte concentrations of 1.0, 2.5, 5.0 and 10 ng/ml and analyzing at least six samples
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per concentration. The LLOQ was defined as the lowest concentration where the relative standard deviation (RSD) as well as the difference of the mean of the calculated concentration from the nominal concentration (bias) was ≤20%. For the evaluation of the linearity of the method, complete calibration curves for blood and urine were analyzed on six consecutive days. The resulting relative peak areas (analyte peak area/IS peak area) were then analyzed using VALISTAT software version 2.00.1 [68] . The Grubbs test was used to check for outliers. No more than one outlier per concentration and two outliers in total for every compound were accepted. Imprecision and accuracy were calculated by analyzing two QC samples of each level on eight consecutive days as required by the GTFCh guidelines [66] . Imprecision was expressed as % RSD of the calculated concentrations and accuracy as the difference of the mean of the calculated concentrations to the nominal concentrations in %. The matrix effect was examined by analyzing blank blood and urine from six different sources with a constant infusion of a solution containing ten analytes selected by retention time (BZP, ethylone, ketamine, MDPPP, MDPV, methcathinone, methylone, naphyrone, norephedrine, pyrovalerone) at a concentration of 1000 ng/ml in mobile phase A into the eluent (10 μl/min flow) by a T-union before it entered the electrospray ion source (postcolumn infusion) [69] . This method for the semiquantitative assessment of matrix effects was originally published by Bonfiglio et al. [70] and its feasibility has also been demonstrated by other researchers [38,71–72] . The extraction efficiency of the method was calculated by comparing the relative peak areas of the analytes of extraction and control samples at concentrations of 30 and 300 ng/ml. The extraction samples (n = 6) were prepared by adding the analytes to blank samples before the extraction step and the IS after the extraction step. In case of the control samples (n = 6) analytes and IS were added after the extraction step. The rest of the sample preparation was carried out as mentioned before. The process of method development and validation is summarized as a flow chart in Figure 1. Results & discussion Method development
A chromatogram of a whole blood QC sample (c = 30 ng/ml) is shown in Figure 2. All analytes eluted between two and 16 min with all isobaric compounds baseline-separated from their isobaric counterparts with the exception of 3-FMC and flephedrone. These two substances are structurally similar and have an
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Detection & quantification of 56 NPS in blood & urine by LC–MS/MS
Research Article
Table 1. MS parameters: precursor ion (Q1), fragment ions (Q3) (quantifier first, qualifiers second and third), expected retention time, declustering potential, entrance potential and collision energy. Collision cell exit potential was 4 V for all transitions. Substance
m/z Q1
m/z Q3
RT (min)
DP (V)
EP (V)
CEP (V)
CE (eV)
2,5-DMA
196.1
179.2/151.1/121.1
10.9
27
7
21
16/22/37
2C-B
260.1
243.0/228.0/213.0
12.9
30
5
14
15/30/41
2C-D
196.2
179.1/ 164.1/149.1
12.0
36
4
8
16/25/35
2C-E
210.2
193.2/ 178.2/163.2
13.8
34
5
21
16/24/38
2C-H
182.0
165.1/ 150.1/135.1
9.0
30
5
22
15/25/39
2C-I
308.1
291.0/ 276.0/261.0
13.9
30
5
18
18/29/40
2C-P
224.1
207.1/ 192.1/163.1
15.1
32
4
24
17/25/37
2C-T-2
242.1
225.1/ 210.1/195.1
13.8
30
4
22
17/27/31
2C-T-4
256.1
239.2/197.1/182.1
14.6
35
4
23
20/28/36
2C-T-7
256.1
239.2/197.1/167.1
15.0
28
4
27
20/29/40
3,4,5-TMA
226.1
209.2/194.2/181.1
9.7
32
4
19
17/25/26
3,4-DMA
196.1
179.1/151.1/107.0
7.8
23
5
19
14/27/50
3-FMC
182.2
164.2/149.1/103.1
5.3
40
5
19
21/29/39
4-MEC
192.1
174.2/144.1
11.1
41
5
19
20/43
4-MTA
182.1
165.1/137.1/117.1
11.8
33
3
18
18/26/27
5-IAI
260.0
116.0/243.0
12.2
45
5
27
38/23
5-MeO-DALT
271.2
110.1/174.1
13.8
36
3
14
19/24
5-MeO-DMT
219.1
174.2/58.0
9.9
29
5
20
21/34
Amphetamine-D5
141.2
93.1
4.9
33
3
16
26
AMT
175.1
158.1/143.1
8.5
22
4
18
16/38
Butylone
222.1
174.2/204.2/146.2
10.4
35
5
22
26/17/34
BZP
177.2
91.1/65.0
3.2
46
4
10
32/62
Cathinone
150.1
132.1/117.1
3.6
36
5
14
19/33
Cocaine-D3
307.2
185.2
13.4
51
5
20
25
Desoxypipradrol
252.2
91.1/167.2
15.1
52
5
9
47/30
DiPT
245.2
144.1/114.1/117.1
13.9
34
6
18
28/20/53
DMPP
191.2
133.1
12.3
55
5
14
35
DMT
189.1
144.1/58.0
8.8
25
5
13
27/29
DOB
274.1
257.1/229.0
13.7
33
4
27
20/28
DOET
224.2
207.1/192.1/177.1
14.6
35
4
24
18/28/37
DOM
210.2
193.1/165.1/178.1
13.1
32
6
20
18/25/27
DPT
245.2
114.1/144.1/86.1
14.4
32
7
14
22/31/38
Ephedrine
166.1
148.1/133.1/115.1
4.1
34
5
18
19/31/39
Ethcathinone
178.1
160.1/130.1/117.1
7.0
40
5
15
18/44/38
2,5-DMA: 2,5-dimethoxyamphetamine; 2C-B: 4-bromo-2,5-dimethoxyphenethylamine; 2C-D: 2,5-dimethoxy-4-methylphenethylamine; 2C-E: 2,5-dimethoxy-4ethylphenethylamine; 2C-H: 2,5-dimethoxyphenethylamine; 2C-I: 2,5-dimethoxy-4-iodophenethylamine; 2C-P: 2,5-dimethoxy-4-n-propylphenethylamine; 2CT-2: 2,5-dimethoxy-4-ethylthiophenethylamine; 2C-T-4: 2,5-dimethoxy-4-isopropylthiophenethylamine; 2C-T-7: 2,5-dimethoxy-4-n-propylthiophenethylamine; 3,4,5-TMA: 3,4,5-trimethoxyamphetamine; 3,4-DMA: 3,4-dimethoxyamphetamine; 4-MEC: 4-methylethcathinone; 4-MTA: 4-methylthioamphetamine; 5-IAI: 5-iodo-2aminoindane; 5-MeO-DALT: N,N-diallyl-5-methoxytryptamine; 5-MeO-DMT: 5-methoxy-N,N-dimethyltryptamine; AMT: α-methyltryptamine; BZP: Benzylpiperazine; CE: Collision energy; CEP: Cell entrance potential; CXP: Cell exit potential; DiPT: N,N-diisopropyltryptamine; DMT: N,N-dimethyltryptamine; DOB: 4-bromo2,5-dimethoxyamphetamine; DOET: 2,5-dimethoxy-4-ethylamphetamine; DOM: 2,5-dimethoxy-4-methylamphetamine; DP: Declustering potential; DPT: N,Ndipropyltryptamine; EP: Entrance potential; mCPP: meta-chlorophenylpiperazine; MDAI: 5,6-methylenedioxy-2-aminoindane; MDBP: Methylenedioxybenzylpiperazine; MDDMA: 3,4-methylenedioxy-N,N-dimethylamphetamine; MDPPP: 3’-4’-methylenedioxy-α-pyrrolidinopropiophenone; MDPV: Methylenedioxypyrovalerone; MeOPP: para-methoxyphenylpiperazine; MiPT: N-methyl-N-isopropyltryptamine; PCP: Phencyclidine; p-fluoro-BZP: para-fluorobenzylpiperazine; PMA: paramethoxyamphetamine, PMMA: para-methoxymethamphetamine; RT: Retention time; TFMPP: 3-trifluoromethylphenylpiperazine; TMA-6: 2,4,6-trimethoxyamphetamine.
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Table 1. MS parameters: precursor ion (Q1), fragment ions (Q3) (quantifier first, qualifiers second and third), expected retention time, declustering potential, entrance potential and collision energy. Collision cell exit potential was 4 V for all transitions (cont.). Substance
m/z Q1
m/z Q3
RT (min)
DP (V)
EP (V)
CEP (V)
CE (eV)
Ethylamphetamine
164.1
91.0/119.0/65.0
9.5
32
6
17
29/17/61
Ethylone
222.1
174.2/204.2 /146.2
9.4
38
5
23
27/21/37
Ethylone-D5
227.2
179.2
9.4
43
5
24
27
Fenfluramine-D10
242.2
161.1
13.2
48
5
20
33
Flephedrone
182.1
164.2/149.1/123.1
5.6
37
5
18
19/30/30
Ketamine
238.1
125.1/220.2
12.1
45
5
21
40/22
Ketamine-D4
242.2
129.0
12.1
42
4
22
38
mCPP
197.1
154.1/118.1
11.7
53
5
21
29/50
MDAI
178.1
161.1/131.1/103.0
5.3
31
5
19
18/26/41
MDBP
221.2
135.1/77.0
4.3
43
5
14
23/57
MDDMA
208.1
163.1/105.1/135.1
10.5
38
6
22
22/37/32
MDEA-D5
213.2
163.1
10.7
38
5
20
19
MDMA-D5
199.1
165.1
9.1
35
5
19
19
MDPPP
248.2
98.1/177.1
11.0
53
4
19
35/23
MDPV
276.2
135.1/126.1
13.8
55
5
25
37/27
MDPV-D8
284.3
134.2
13.8
57
5
22
38
MeOPP
193.1
119.1/150.1
7.8
51
5
20
35/27
Mephedrone
178.1
145.1/160.1 91.1
9.6
36
4
18
28/18/47
Mephedrone-D3
181.1
163.1
9.6
37
8
19
16
Methcathinone
164.1
146.1/130.1/105.1
4.9
38
5
18
19/43/32
Methedrone
194.1
176.2/161.1
8.9
35
5
21
21/28
Methylone
208.1
160.1/132.1/190.1
7.3
31
5
21
24/37/17
MiPT
217.2
86.1/144.1
12.0
28
7
12
18/25
Naphyrone
282.2
141.1/211.1
15.6
57
5
21
37/23
Norephedrine
152.1
134.1/117.1
2.7
29
3
16
15/25
PCP
244.2
91.1/159.2
15.3
29
3.5
22
51/19
PCP-D5
249.2
164.2
15.2
34
4
22
22
p-Fluoro-BZP
195.1
109.0/83.0
4.4
49
5
20
33/64
PMA
166.1
149.1/121.0/91.1
7.8
26
5
19
14/25/44
PMMA
180.2
149.2/121.0
9.6
30
5
20
19/29
Pyrovalerone
246.2
105.1/175.2
14.5
44
5
24
32/24
TFMPP
231.1
188.1/118.1
12.7
51
5
15
30/57
TMA-6
226.1
209.2/181.1/121.1
13.0
24
4
23
18/28/39
2,5-DMA: 2,5-dimethoxyamphetamine; 2C-B: 4-bromo-2,5-dimethoxyphenethylamine; 2C-D: 2,5-dimethoxy-4-methylphenethylamine; 2C-E: 2,5-dimethoxy-4ethylphenethylamine; 2C-H: 2,5-dimethoxyphenethylamine; 2C-I: 2,5-dimethoxy-4-iodophenethylamine; 2C-P: 2,5-dimethoxy-4-n-propylphenethylamine; 2CT-2: 2,5-dimethoxy-4-ethylthiophenethylamine; 2C-T-4: 2,5-dimethoxy-4-isopropylthiophenethylamine; 2C-T-7: 2,5-dimethoxy-4-n-propylthiophenethylamine; 3,4,5-TMA: 3,4,5-trimethoxyamphetamine; 3,4-DMA: 3,4-dimethoxyamphetamine; 4-MEC: 4-methylethcathinone; 4-MTA: 4-methylthioamphetamine; 5-IAI: 5-iodo-2aminoindane; 5-MeO-DALT: N,N-diallyl-5-methoxytryptamine; 5-MeO-DMT: 5-methoxy-N,N-dimethyltryptamine; AMT: α-methyltryptamine; BZP: Benzylpiperazine; CE: Collision energy; CEP: Cell entrance potential; CXP: Cell exit potential; DiPT: N,N-diisopropyltryptamine; DMT: N,N-dimethyltryptamine; DOB: 4-bromo2,5-dimethoxyamphetamine; DOET: 2,5-dimethoxy-4-ethylamphetamine; DOM: 2,5-dimethoxy-4-methylamphetamine; DP: Declustering potential; DPT: N,Ndipropyltryptamine; EP: Entrance potential; mCPP: meta-chlorophenylpiperazine; MDAI: 5,6-methylenedioxy-2-aminoindane; MDBP: Methylenedioxybenzylpiperazine; MDDMA: 3,4-methylenedioxy-N,N-dimethylamphetamine; MDPPP: 3’-4’-methylenedioxy-α-pyrrolidinopropiophenone; MDPV: Methylenedioxypyrovalerone; MeOPP: para-methoxyphenylpiperazine; MiPT: N-methyl-N-isopropyltryptamine; PCP: Phencyclidine; p-fluoro-BZP: para-fluorobenzylpiperazine; PMA: paramethoxyamphetamine, PMMA: para-methoxymethamphetamine; RT: Retention time; TFMPP: 3-trifluoromethylphenylpiperazine; TMA-6: 2,4,6-trimethoxyamphetamine.
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Bioanalysis (2015) 7(9)
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Detection & quantification of 56 NPS in blood & urine by LC–MS/MS
Research Article
Full Scan Q1 Expected [M + H]+ ± 50 amu
Determination of m/z of [M + H]+
EPI spectra (CE 20, 35, 50 eV + CES 35 ± 15 eV)
Spectra for MS/MS library
Optimisation of MRM parameters for 3–4 fragments
Quantitative (s)MRM method 2–3 MRMs per compound
Addition of new compounds influences previous validation, revalidation necessary
New NPS compound
Sample preparation
Validation All validation criteria fulfilled Quantitative
Not all validation criteria fulfilled Semiquantitative
Figure 1. Method development and validation process. Enhanced product ion spectra, acquired at three different collision energies are collected in a database [73] . EPI: Enhanced product ion spectra; NPS: New psychoactive substances; (s)MRM: (scheduled) Multiple reaction monitoring.
almost identical fragmentation pattern. Flephedrone and 3-FMC are not completely baseline-separated (chromatographic resolution, RS ≈ 1.4), but still can be distinguished by the difference of their retention times. The lack of full baseline-separation allows only for semiquantitative detection of these two compounds. A target scan time of 1 s was chosen to ensure a minimum dwell time of at least 15 ms for each transition for optimal sensitivity. At the lowest calibrator, peak width is at least ∼9 s. Therefore, a cycle time of 1 s is sufficient to provide enough data points for proper peak integration. With newer, more sensitive instruments a shorter cycle time might be chosen. Validation
Testing for selectivity and specificity by analyzing blank samples from different sources and solutions of single analytes, respectively, revealed no significant interference from matrix components or cross-talk between different analytes. Ion ratios have been
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summarized in Supplementary Table 1. LLOQs were generally between 1.0 and 10 ng/ml for both matrices. Values for every analyte are listed in Table 2. Calibration curves were linear up to 1000 ng/ml for all analytes (weighting: 1/x, R 2 > 0.99 for all analytes). Regression parameters for all analytes are presented in Supplementary Table 1. Detailed data for imprecision and accuracy are also listed in Table 2. In general, all analytes matched the validation criteria of RSD ≤ 15% and bias ≤ 15% for both QC samples, except for the substances given in Table 3. These compounds can therefore only be analyzed semiquantitatively. Ion suppression was assessed qualitatively by postcolumn infusion. Ten different compounds were chosen for this experiment based on their retention time. Comparison of different analytes shows very similar results on a semiquantitative level. Therefore, analogous results can be expected for the remaining analytes. Exact quantification of matrix effects, following, i.e., the
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Research Article Ambach, Hernández Redondo, König, Angerer, Schürch & Weinmann
B
5.9e5
2.0e5 1.8e5
5.0e5
1.6e5
4.0e5
Intensity (cps)
Intensity (cps)
A
3.0e5 2.0e5 10.87
1.0e5 8.0e4 6.0e4
5
10
15
0.0
20
Time (min)
D 9.0e5
3.0e5
7.0e5
10
15
20
Intensity (cps)
8.0e5
2.5e5 2.0e5 5.16
1.0e5
5.50
6.0e5 5.0e5 4.0e5 3.0e5 13.81
2.0e5
5.0e4 0.0
5
Time (min)
3.8e5 3.5e5
1.5e5
12.83
2.0e4 11.06
C
Intensity (cps)
1.2e5
4.0e4
1.0e5 0.0
1.4e5
1.0e5 5
10
15
20
0.0
Time (min)
5
10
15
20
Time (min)
Figure 2. Extracted ion chromatograms of a whole blood QC sample, c = 30 ng/ml. (A) amphetamines; (B) phenethylamines and aminoindanes; (C) cathinones; (D) piperazines, tryptamines, dissociatives and others.
method of Matuszewski [74] , can be somewhat misleading in the eyes of the authors, as matrix effects vary from sample to sample and the validation process cannot account for all interindividual variances in matrix composition. The only way to really compensate for matrix effects would be the quantification by standard addition (repeated analysis of sample after spiking with different concentrations of the analyte of interest and subsequent back calculation). For whole blood samples (Figure 3A) some periodic ion suppression was seen between 10 and 15 min, presumably by lipophilic matrix compounds. However, there seems to be little to no correlation with the retention times of the compounds listed in Table 3, so this ion suppression appears not to have a significant effect on reproducibility. For urine (Figure 3B), there is no significant matrix effect compared with a solvent blank (Figure 3C). Extraction efficiencies were between 60 and 86% in whole blood and between 57 and 97% in urine. Detailed data is presented in Table 2.
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Bioanalysis (2015) 7(9)
As far as we are aware, most published methods for the detection of NPS focus on single substance groups such as cathinones [42–43,45,51,53,64] , tryptamines [65] , piperazines [41,46] or 2C/DOX substances [44] . Methods covering several substance classes include fewer substances than the method presented here [47,52,58,62] or are intended for qualitative screening purposes rather than quantification [40,42,50,60,64] . More comprehensive methods have been developed by Al-Saffar et al. (27 compounds, urine) [38] , Paul et al. (35 compounds, urine) [39] and Swortwood et al. (32 substances, serum) [49] . Therefore, the presented method for the sensitive detection and quantification of 56 NPS across eight different substance groups in both urine and whole blood samples is a meaningful addition to the collection of tools currently available to forensic and clinical toxicologists. Metabolites & urinalysis
When analyzing urine samples, possible metabolization of the parent analytes should generally be considered.
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Detection & quantification of 56 NPS in blood & urine by LC–MS/MS
Research Article
% Extraction efficiency
% Bias†
Table 2. Validation results. Substance
LLOQ (ng/ml) Blood Urine
Blood
Urine
% RSD Blood
Urine
Blood
Urine
QC 1
QC 2
QC 1
QC 2
QC 1
QC 2
QC 1
QC 2
2,5-DMA
5.00
2.50
79
89
11
9
11
8
7
-13
13
-14
2C-B
2.50
2.50
80
89
11
13
14
13
15
6
6
-6
2C-D
5.00
2.50
86
87
8
8
11
9
-1
-9
9
-12
2C-E
2.50
2.50
85
89
13
14
17
17
27
0
36
-1
2C-H
2.50
2.50
77
79
10
15
12
13
18
3
4
-4
2C-I
2.50
2.50
82
90
8
13
13
14
22
-4
33
-3
2C-P
10.0
2.50
78
91
8
8
10
9
12
-12
12
-17
2C-T-2
2.50
10.0
73
92
12
10
12
15
37
-14
7
-22
2C-T-4
2.50
1.00
72
92
11
11
11
9
0
-2
-14
-12
2C-T-7
2.50
5.00
70
92
14
12
16
13
24
4
-4
-12
3,4,5-TMA
5.00
2.50
82
67
9
15
13
11
6
4
3
-7
3,4-DMA
1.00
2.50
79
61
12
14
12
15
-7
-12
4
-6
4-MEC
5.00
1.00
79
83
10
12
12
12
2
2
-8
-11
4-MTA
2.50
5.00
67
96
14
13
18
22
11
-4
-3
0
5-IAI
5.00
2.50
76
97
14
14
16
13
14
-1
8
-6
5-MeO-DALT
2.50
1.00
66
90
14
11
12
11
2
-3
-21
-16
5-MeO-DMT
2.50
1.00
64
94
11
8
8
6
-13
-14
-6
-13
AMT
2.50
2.50
65
88
12
9
7
9
-14
-16
-15
-12
Butylone
5.00
2.50
78
88
9
11
13
8
6
-3
12
-6
BZP
2.50
2.50
80
77
10
9
9
9
-7
-9
-9
-12
Cathinone
2.50
2.50
79
58
5
12
11
9
1
1
-9
-10
Desoxypipradrol
2.50
1.00
77
91
8
7
9
8
-4
-9
-7
-16
DiPT
2.50
1.00
64
92
13
12
9
16
-11
-13
-10
-16
DMT
5.00
2.50
69
95
12
10
11
11
12
3
-1
-7
DOB
5.00
2.50
81
94
13
11
11
9
-1
-11
0
-11
DOET
2.50
1.00
79
89
8
10
12
12
30
-5
-5
-12
DOM
2.50
2.50
79
94
5
8
9
8
8
-8
9
-11
DPT
2.50
1.00
63
92
10
7
6
8
-6
-12
-10
-15
Ephedrine
2.50
2.50
79
70
10
11
9
12
-5
-11
-7
-12
Ethcathinone
2.50
2.50
75
72
10
11
9
12
-8
-12
-10
-8
%bias: relative difference of mean from nominal value in %. 3-FMC and flephedrone were not baseline-separated and determined only semiquantitatively. Therefore, no validation data is available for these substances. 2,5-DMA: 2,5-dimethoxyamphetamine; 2C-B: 4-bromo-2,5-dimethoxyphenethylamine; 2C-D: 2,5-dimethoxy-4-methylphenethylamine; 2C-E: 2,5-dimethoxy4-ethylphenethylamine; 2C-H: 2,5-dimethoxyphenethylamine; 2C-I: 2,5-dimethoxy-4-iodophenethylamine; 2C-P: 2,5-dimethoxy-4-n-propylphenethylamine; 2C-T-2: 2,5-dimethoxy-4-ethylthiophenethylamine; 2C-T-4: 2,5-dimethoxy-4-isopropylthiophenethylamine; 2C-T-7: 2,5-dimethoxy-4-n-propylthiophenethylamine; 3,4,5-TMA: 3,4,5-trimethoxyamphetamine; 3,4-DMA: 3,4-dimethoxyamphetamine; 4-MEC: 4-methylethcathinone; 4-MTA: 4-methylthioamphetamine; 5-IAI: 5-iodo-2-aminoindane; 5-MeO-DALT: N,N-diallyl-5-methoxytryptamine; 5-MeO-DMT: 5-methoxy-N,N-dimethyltryptamine; AMT: α-methyltryptamine; BZP: benzylpiperazine; DiPT: N,N-diisopropyltryptamine; DMT: N,N-dimethyltryptamine; DOB: 4-bromo-2,5-dimethoxyamphetamine; DOET: 2,5-dimethoxy-4ethylamphetamine; DOM: 2,5-dimethoxy-4-methylamphetamine; DPT: N,N-dipropyltryptamine; mCPP: meta-chlorophenylpiperazine; MDAI: 5,6-methylenedioxy2-aminoindane; MDBP: methylenedioxybenzylpiperazine; MDDMA: 3,4-methylenedioxy-N,N-dimethylamphetamine; MDPPP: 3’-4’-methylenedioxyα-pyrrolidinopropiophenone; MDPV: methylenedioxypyrovalerone; MeOPP: para-methoxyphenylpiperazine; MiPT: N-methyl-N-isopropyltryptamine; PCP: phencyclidine; p-fluoro-BZP: para-fluorobenzylpiperazine; PMA: para-methoxyamphetamine, PMMA: para-methoxymethamphetamine; TFMPP: 3-trifluoromethylphenylpiperazine; TMA-6: 2,4,6-trimethoxyamphetamine. †
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1127
Research Article Ambach, Hernández Redondo, König, Angerer, Schürch & Weinmann
Table 2. Validation results (cont.). Substance
LLOQ (ng/ml)
% Extraction efficiency
% RSD Blood
% Bias†
Blood Urine
Blood
Urine
QC 1
QC 2
QC 1
Urine QC 2
QC 1
Blood QC 2
QC 1
Urine QC 2
Ethylamphetamine 2.50
2.50
82
89
11
13
14
11
9
5
11
1
Ethylone
2.50
1.00
79
91
7
8
8
7
1
-11
-2
-12
Ketamine
2.50
1.00
78
95
12
7
8
6
-7
-15
-5
-14
mCPP
2.50
2.50
80
95
11
13
11
10
14
3
9
-4
MDAI
1.00
2.50
77
91
10
11
8
10
-5
-20
-5
-10
MDBP
2.50
2.50
76
86
10
10
10
11
-13
-12
-5
-9
MDDMA
2.50
2.50
75
92
9
12
9
9
1
-9
0
-8
MDPPP
2.50
1.00
80
93
12
10
9
7
2
-2
-5
-14
MDPV
5.00
2.50
82
96
7
10
8
7
9
-5
4
-11
MeOPP
2.50
1.00
60
80
12
11
12
6
4
-3
-10
-17
Mephedrone
2.50
2.50
81
81
8
13
13
10
5
4
6
-2
Methcathinone
2.50
1.00
81
73
8
11
12
11
13
7
-7
-7
Methedrone
2.50
2.50
80
82
7
11
14
11
0
-1
6
-4
Methylone
2.50
1.00
78
84
8
11
11
11
2
3
-8
-8
MiPT
2.50
2.50
65
94
9
8
9
8
-8
-14
-4
-13
Naphyrone
2.50
1.00
77
87
7
9
9
10
4
-6
-12
-14
Norephedrine
2.50
5.00
77
57
9
14
15
13
17
-4
-2
-5
PCP
2.50
2.50
81
90
6
10
8
7
12
-1
1
-7
p-Fluoro-BZP
2.50
2.50
76
86
13
8
7
12
-2
-15
-5
-15
PMA
2.50
1.00
79
88
8
10
11
11
2
-11
-9
-14
PMMA
2.50
2.50
78
90
7
8
7
8
11
-17
6
-13
Pyrovalerone
2.50
1.00
76
90
6
10
12
10
-1
-2
-7
-11
TFMPP
2.50
1.00
73
96
10
10
10
9
-15
-6
-11
-8
TMA-6
5.00
2.50
78
78
8
10
9
9
-1
-3
2
-9
%bias: relative difference of mean from nominal value in %. 3-FMC and flephedrone were not baseline-separated and determined only semiquantitatively. Therefore, no validation data is available for these substances. 2,5-DMA: 2,5-dimethoxyamphetamine; 2C-B: 4-bromo-2,5-dimethoxyphenethylamine; 2C-D: 2,5-dimethoxy-4-methylphenethylamine; 2C-E: 2,5-dimethoxy4-ethylphenethylamine; 2C-H: 2,5-dimethoxyphenethylamine; 2C-I: 2,5-dimethoxy-4-iodophenethylamine; 2C-P: 2,5-dimethoxy-4-n-propylphenethylamine; 2C-T-2: 2,5-dimethoxy-4-ethylthiophenethylamine; 2C-T-4: 2,5-dimethoxy-4-isopropylthiophenethylamine; 2C-T-7: 2,5-dimethoxy-4-n-propylthiophenethylamine; 3,4,5-TMA: 3,4,5-trimethoxyamphetamine; 3,4-DMA: 3,4-dimethoxyamphetamine; 4-MEC: 4-methylethcathinone; 4-MTA: 4-methylthioamphetamine; 5-IAI: 5-iodo-2-aminoindane; 5-MeO-DALT: N,N-diallyl-5-methoxytryptamine; 5-MeO-DMT: 5-methoxy-N,N-dimethyltryptamine; AMT: α-methyltryptamine; BZP: benzylpiperazine; DiPT: N,N-diisopropyltryptamine; DMT: N,N-dimethyltryptamine; DOB: 4-bromo-2,5-dimethoxyamphetamine; DOET: 2,5-dimethoxy-4ethylamphetamine; DOM: 2,5-dimethoxy-4-methylamphetamine; DPT: N,N-dipropyltryptamine; mCPP: meta-chlorophenylpiperazine; MDAI: 5,6-methylenedioxy2-aminoindane; MDBP: methylenedioxybenzylpiperazine; MDDMA: 3,4-methylenedioxy-N,N-dimethylamphetamine; MDPPP: 3’-4’-methylenedioxyα-pyrrolidinopropiophenone; MDPV: methylenedioxypyrovalerone; MeOPP: para-methoxyphenylpiperazine; MiPT: N-methyl-N-isopropyltryptamine; PCP: phencyclidine; p-fluoro-BZP: para-fluorobenzylpiperazine; PMA: para-methoxyamphetamine, PMMA: para-methoxymethamphetamine; TFMPP: 3-trifluoromethylphenylpiperazine; TMA-6: 2,4,6-trimethoxyamphetamine. †
While synthetic cannabinoids show extensive metabolism to the point where most of the time only metabolites are detectable in urine samples, cathinones are metabolized to a far lesser degree [75] . In the case of cathinones and piperazines, parent compounds are generally abundant in urine, for piperazines even in higher concentrations than their respective metabolites [76] . A reported intoxication with 5-MeO-DiPT, where significant concentrations of the parent
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Bioanalysis (2015) 7(9)
compound (229 ng/ml) were present in urine [62] , indicates that this is also true for tryptamines. Therefore, focusing on parent compounds should be sufficient for the detection of stimulant NPS in urine, especially as there are still little pharmacokinetic data available for such compounds and even less reference standards for the quantification of their metabolites. It should be self-evident, however, that the inclusion of metabolites, such as the corresponding ephedrines for
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Detection & quantification of 56 NPS in blood & urine by LC–MS/MS
Research Article
Table 3. Substances that can only be determined semiquantitatively. Semiquantitatively in urine
Semiquantitatively in whole blood Semiquantitatively in both matrices
2C-P
2C-H
2C-E
4-MTA
AMT
2C-I
5-IAI
DOET
2C-T-2
5-MeO-DALT
MDAI
2C-T-7
Desoxypipradrol
Norephedrine
3-FMC
DiPT
PMMA
Flephedrone
MeOPP
cathinones, may further improve the detection of NPS consumption. Unfortunately, at the time of method development, such substances were not available to us as reference standards. Another issue that should be taken into account is the fact that some of the parent compounds themselves may be metabolites of other substances. Ephedrine and Norephedrine can be formed by either metabolic reduction of methcathinone and cathinone, respectively, or oxidative metabolization of methamphetamine and amphetamine, respectively. Piperazine derivatives may be formed from several pharmaceutical drugs: mCPP is a known metabolite of trazodone, MDBP may be formed from piribedil or fipexide while BZP is a metabolite of befuraline and piberaline. 2C compounds may be formed from their NBOMe derivatives by N-dealkylation. Parent compounds and metabolites may be detectable for a longer timeframe in urine compared with blood samples. Analysis of blood samples
Although concentrations of active compounds may be lower in blood than in urine, in the forensic field impairment due to drug consumption needs to be assessed. This is usually underpinned by concentrations of the active compound in blood. In postmortem cases with suspected intoxications, analysis of blood samples is important to determine the cause of death; urine – if available – is used for screening only. Depending on the jurisdiction, urine may only be used for preliminary testing by the police while blood is sent to a forensic laboratory for confirmatory analysis. Application to authentic samples
The developed method has also been applied to authentic samples and some positive results are presented in Table 4. In these cases the most common substance is cathinone. In almost all cases (except case 1) parent compounds are present in both whole blood and urine. Norephedrine is a metabolite of cathinone generated by a reduction of the keto group to a hydroxyl group and
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is also present in all cases involving cathinone (cases 2–5, 8). In an analogous fashion ephedrine is a metabolite of methcathinone (case 8). Norephedrine can also appear as an oxidative metabolite of amphetamine (cases 1 and 7). In cases 1 and 5 ketamine was detected. The first case involved a traffic control following suspicious driving behavior indicating a recreational use of ketamine. The second case on the other hand involved a heavy traffic accident where administration of ketamine by paramedics or emergency physicians is a plausible scenario although no information in this regard was supplied by police. Continued method development
One of the distinctive characteristics of the NPS market is its ever-changing nature. Compounds that are included into the narcotics legislation often decline/ disappear from the markets (with the exception of a few compounds) and new substances are introduced as their replacements [77] . Therefore, method adaption and expansion is a necessity. Being MRM-based, the presented method can easily be modified by editing the list of MRM transitions. This, however, poses the question of revalidation. L Huber explicitly suggests method revalidation when ‘[n]ew compounds are analyzed that are not within the method’s original scope’ [78] and the SWGTOX guidelines [79] recommend revalidating the parameters that are affected by the change to the method. In the case of including new analytes this would obviously entail revalidating selectivity and specificity for all compounds. Moreover, if a regular MRM approach is used, additional compounds will increase the cycle time which in turn might result in less data points per peak and therefore deteriorating peak shape. Thus, reproducibility might be negatively affected and RSD and bias should be re-evaluated for all compounds. In case of an sMRM method with a set cycle time, the addition of new analytes will lower the dwell times for compounds that also elute in the new compounds’ retention time window. This can potentially affect sensitivity.
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1129
Research Article Ambach, Hernández Redondo, König, Angerer, Schürch & Weinmann Therefore, the LODs and/or LLOQs for all compounds with retention time windows overlapping with the new analytes would need to be revalidated. Since these criteria do not have to be checked for all
analytes, the use of sMRM might be more efficient for method expansion. Naturally, for the new substances all validation criteria need to be checked. Expanding and subsequently revalidating a multianalyte method
A 1.4e5
1.2e5
Intensity (cps)
1.0e5
8.0e4
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2.0e4 0 .3 0 0.63
2.19
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6.59 6.83 7.71 7 .9 4 9.22
9.73
5
10.23
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17.71 17.92
18.35
15
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Time (min) B 1.4e5
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3.39
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4.184.89
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10.12
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11.70
12.89
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14.32
0.0 5
10
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15.21
15.94
17.95
18.31
19.70
20
Time (min) Figure 3. Test for matrix effects. Ion suppression by postcolumn infusion in (A) whole blood samples, (B) urine samples and (C) solvent blank samples.
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Bioanalysis (2015) 7(9)
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Detection & quantification of 56 NPS in blood & urine by LC–MS/MS
Research Article
C 2.0e5 1.8e5 1.6e5
Intensity (cps)
1.4e5 1.2e5 1.0e5 8.0e4 6.0e4 4.0e4 2.0e4 0.0 5
10
15
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Time (min) Figure 3. Test for matrix effects (cont.). Ion suppression by postcolumn infusion in (A) whole blood samples, (B) urine samples and (C) solvent blank samples (cont.).
might consequently prove to be a rather time consuming effort depending on the number of analytes and whether MRM or sMRM is used. The minimum of revalidation should include the following: Linearity of concentration-response
relationship should be investigated in the expected concentration range of the new analytes. Specificity and selectivity should be confirmed by analyzing different blank matrix samples and samples spiked with the previously included analytes (e.g., old calibrators
Table 4. New psychoactive substances found in matching urine and whole blood samples. Sample (n)
NPS in urine
NPS in whole blood
1
Ketamine, norephedrine
Amphetamine (
