Two immunoassays for the detection of 2C-B and related hallucinogenic phenethylamines

Accepted Manuscript

Two immunoassays for the detection of 2C-B and related

hallucinogenic phenethylamines

Anna Šuláková, Lucie Fojtíková, Barbora Holubová, Kateřina

Bártová, Oldřich Lapčík, Martin Kuchař

PII:

DOI:

Reference:

S1056-8719(18)30733-0

https://doi.org/10.1016/j.vascn.2018.11.001

JPM 6543

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Journal of Pharmacological and Toxicological Methods

Received date:

Revised date:

Accepted date:

6 September 2018

26 October 2018

21 November 2018

Please cite this article as: Anna Šuláková, Lucie Fojtíková, Barbora Holubová, Kateřina

Bártová, Oldřich Lapčík, Martin Kuchař , Two immunoassays for the detection of 2C-

B and related hallucinogenic phenethylamines. Jpm (2018), https://doi.org/10.1016/

j.vascn.2018.11.001

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ACCEPTED MANUSCRIPT

Two immunoassays for the detection of 2C-B and related hallucinogenic

phenethylamines

Anna Šuláková^a,b,danna.sulakova@nudz.cz, Lucie Fojtíková^a,clucie.fojtikova@vscht.cz,

Barbora Holubová^cbarbora.holubova@vscht.cz, Kateřina Bártová^a,b, Oldřich Lapčík^a

oldrich.lapcik@vscht.cz, Martin Kuchař^a,b,d,*martin.kuchar@vscht.cz

^aDepartment of Chemistry of Natural Compounds, University of Chemistry and Technology

Prague, Technická 5, 166 28 Praha 6 – Dejvice, Czechia

^bForensic Laboratory of Biologically Active Substances, University of Chemistry and

Technology Prague, Technická 3, 166 28 Praha 6 – Dejvice, Czechia

^cDepartment of Biochemistry and Microbiology, University of Chemistry and Technology

Prague, Technická 3, 166 28 Praha 6 – Dejvice, Czechia

^dDepartment of Experimental Neurobiology, National Institute of Mental Health, Topolová

748, 250 67 Klecany, Czechia

Corresponding author at: University of Chemistry and Technology Prague, Technická 3, 166

28 Praha 6 – Dejvice, Czechia.

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Abstract

Introduction

The use of new psychoactive subtances as drugs of abuse has dramatically increased over the

last years. Hallucinogenic phenethylamines gained particular popularity as they have both

stimulating and psychedelic effects. Although generally perceived as safe, these illicit drugs

pose a serious health risk; they have been linked to cases of severe poisoning or even deaths.

Therefore, simple, cost-effective and reliable methods are needed for rapid determination of

abused hallucinogens.

Methods

For this purpose, two haptens derived from 2C-H were designed, synthesized and

subsequently attached to a carrier protein. Polyclonal antibodies obtained from a rabbit

immunized with one of the prepared immunogens were used for the development of two

immunoassays.

Results

In this study, a lateral flow immunoassay (LFIA) and an enzyme linked immunosorbent assay

(ELISA) for the detection of 2C-B and related hallucinogenic phenethylamines in urine were

developed. The presented LFIA is primarily suitable for on-site monitoring as it is simple and

can provide a visual evidence of 2C-B presence within a few minutes. Its reasonable

sensitivity (LOD_LFIAꢀ=ꢀ15 ± 7 ng mL⁻¹) allows detection of the drug presence in urine after

acute exposure. For greater accuracy, highly sensitive ELISA (LOD_ELISAꢀ=ꢀ6 ± 3 pg mL⁻¹) is

proposed for toxicological quantitative analyses of positive samples captured by the LFIA.

Discussion

The comparison of the ELISA with the well-established UHPLC-MS-MS method shows

excellent agreement of results, which confirms good potential of the ELISA to be used for

routine analyses of 2C-B and related hallucinogenic phenethylamines of both main sub-

families.

Keywords: 2C-B, ELISA, hapten synthesis, LFIA, methods, polyclonal antibodies

production,

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1. Introduction

New psychoactive substances (NPS) have expanded throughout illicit drug market over the

last decades. By the beginning of 2018, 670 new abused substances have been identified in

Europe and their number is still growing (EMCDDA, 2018). The increasing popularity of

these substances is attributed to their easy accessibility (especially via online shops), low cost,

non-invasive routes of administration and users’ conviction that they are safe for recreational

use.

Hallucinogenic phenethylamines have become particularly popular as they have stimulating

and psychedelic effects, produce euphoria, increased empathy and sociability, as well as

perceptual enhancement (Shulgin et al., 1991; Pasin et al., 2017; Zawilska, 2015). They can

be divided into two main groups. The 2C series drugs are ring substituted phenethylamines,

which consist of β-phenethylamine backbone, two methoxy groups in the positions 2 and 5

and a hydrophobic substituent in the position 4. These features, essential for hallucinogenic

activity, are shared by the DO series drugs, ring substituted amphetamines, which differ only

in the presence of a methyl group on the α-carbon (Shulgin et al., 1991; Theobald et al., 2006;

Monte et al., 1996). Drugs of both series bind to 5-HT₂receptors and act as their agonists

(Theobald et al., 2006; de Boer et al., 2004). Following intoxication with 2C series drugs,

pathological neuropsychiatric manifestations have been described (Bosak et al., 2013; Drees

et al., 2009; Miyajima et al., 2008) and also a few fatal intoxications have been attributed to

these hallucinogens (Curtis et al., 2003; Sacks et al., 2012; Topeff et al., 2011).

2,5-Dimethoxy-4-bromophenethylamine

(2C-B)

is

a

prominent

hallucinogenic

phenethylamine. It was first synthesized in the mid-1970s by Alexander Shulgin and it was

originally designed for the purposes of psychological research (Shulgin et al., 1975). The

substance was later sold legally as remedy for impotence and frigidity (Kerrigan et al., 2011)

and, eventually, started to be abused as a psychoactive drug. It was found in drug seizures in

UK and USA over 25 years ago (King, 2013; Ragan et al., 1985) and 2C-B has been

consistently accessible to users since then. Although it was listed as a schedule II substance, it

is still one of the most widespread NPS in Europe (King, 2013; Hondebrink et al., 2015). It is

particularly popular due to short duration and mild nature of the intoxication state (Shulgin et

al., 1975; Ragan et al., 1985; González et al., 2015). No fatalities caused by 2C-B overdose

have been reported to date. However, the drug can pose a serious health risk to vulnerable

individuals, as indicated by variable susceptibility to toxic effects of 2C-B in human

population described in the literature (Carmo et al., 2005). A case report of induced psychosis

after ingestion of a single tablet of 2C-B (Huang et al., 2011), cerebral vasculopathy (Ambrose

et al., 2010) and repeated generalized tonic-clonic seizures with urinary incontinence after

laboratory confirmed 2C-B ingestion (Ho et al., 2013) were all published in scientific

literature. These descriptions of severe neurological and psychopathological consequences of

2C-B disprove the beliefs that it is a safe drug.

Studies on metabolism of 2C-B revealed that the majority of the drug undergoes

biotransformation. Two main metabolic pathways exist. In the first one, 2C-B is deaminated

to the corresponding aldehyde and subsequently oxidized to carboxylic acid or reduced to

alcohol metabolites. The second metabolic pathway leads to the formation of O-desmethyl

derivatives, in which the amino groups are acylated (Carmo et al., 2005; Kanamori et al.,

2003; Kanamori et al., 2005). Although extensively metabolized, a significant amount of the

parent compound is excreted unchanged in urine, especially during the first few hours after

administration (Carmo et al., 2004; de Boer et al., 1999; Kanamori et al., 2002; Kanamori et

al., 2013). This is of high importance, as presence of the drug in urine can be used to identify

acute exposure to 2C-B.

Several analytical methods have been applied for the determination of substituted

phenethylamines in urine or other biological matrices. Most frequently, they are based on high

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performance liquid chromatography (Bogusz et al., 2000; Vorce et al., 2004; Nordgren et al.,

2005; Pichini et al., 2008; Wohlfarth et al., 2010; Kerrigan et al., 2014) or gas

chromatography (Vorce et al., 2004; Namera et al., 2002; Habrdova et al., 2005; Ishida et al.,

2006; Kim et al., 2007) coupled to various selective detectors, but capillary electrophoretic

techniques (Boatto et al., 2005; Tsai et al., 2006) have also been described. Chromatographic

methods provide specific and sensitive analysis, but they require highly trained operators and

sophisticated equipment. Moreover, they are not suitable for screening analyses, as they

usually require sample preparation, are relatively expensive and time-consuming.

Clinical and forensic toxicology laboratories often utilize immunoassays as rapid and cost-

effective analytical methods for the determination of drug content, especially when large

numbers of samples are tested. The possibility of analyzing liquid samples or solid sample

extracts without any purification is one of the most outstanding advantages the immunoassays

have over instrumental methods. Several immunoassays directed toward amphetamine-like

psychostimulants were examined for the detection of 2C-B. Unfortunately, phenethylamines

of the 2C series did not cross-react with the commercial immunoassays (Kerrigan et al., 2011;

Apollonio et al., 2007; Nakanishi et al., 2012; Swortwood et al., 2014), the only exception

being EMIT II Plus Amphetamines assays (Siemens) (Petrie et al., 2013). Recently,

production of antibodies targeted separately at 2C and DO sub-families of phenethylamines

was described in patent literature (Benchikh et al., 2018), but a critical need for a simple,

class-based method for rapid determination of hallucinogenic phenethylamines remains.

Therefore, our aim was to develop both reasonably sensitive lateral flow immunoassay

(LFIA), that would be as simple as possible to be applied by the state authorities for rapid

roadside and on-site monitoring of 2C-B in urine samples or extracts from confiscated tablets,

and also enzyme-linked immunosorbent assay (ELISA) for toxicological quantitative analyses

of positive samples captured by the aforementioned LFIA. To the best of our knowledge,

these are the first immunoassays capable to detect hallucinogenic phenethylamines of both

sub-families.

2. Materials and methods

2.1. Materials and reagents

Goat anti-rabbit antibody (GAR), donkey anti-rabbit antibody (DAG) and peroxidase labelled

goat anti-rabbit antibody (GAR-Po) were from Nordic Immunological Laboratories,

Netherlands. Gold colloid nanoparticles (an average diameter of 40 nm) were purchased from

BB international, UK. Bovine serum albumin (BSA), Aerosol-OT (AOT, bis(2-

ethylhexyl)sulfosuccinate),

Tween

20,

sucrose,

polyethylene

glycol

(PEG),

2,5-dimethoxybenzaldehyde,

nitromethane,

titanium

tetrachloride,

methyl

4-chloro-4-oxobutyrate, ethyl chlorooxoacetate, 10 % palladium on carbon and N-

hydroxysuccinimide (NHS) were purchased from Sigma-Aldrich Inc., USA. Surfactant 10G

was from Fitzgerald Industries International, USA. N,N’-Dicyclohexylcarbodiimide (DCC)

was obtained from Fluka, Switzerland. N,N-dimethylformamide (DMF) extra dry was

purchased from Acros Organics, Belgium. All the other solvents were obtained from Penta,

Czechia. All the reagents were used as supplied, unless otherwise indicated. Thin layer

chromatography (TLC) was performed on Merck aluminium backed sheets coated with 60F

254 silica gel. Nitrocellulose membranes (PRIMA 85; AE 98; AE 99; AE 100) were from

Whatman GmbH, Germany. Other nitrocellulose membranes (HiFlow Plus HFB13504;

HiFlow Plus HFB135UB; HiFlow Plus HFC18002), laminated card (HF000MC100), glass

fiber conjugate pad (CFCP03000), cellulose fiber sample pad (CFSP173000) and absorbent

pad (CFSP) were supplied from Millipore Corp., USA. 96-Well polystyrene microtiter plates

Costar 9018 were purchased from Corning Inc., USA.

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2C-B standard and all the other drug standards used for cross-reactivity studies were obtained

from Alfarma s.r.o., Czechia or Cayman Pharma, Czechia. Individual stock standard solutions

containing 1 mg mL^-1of each compound were prepared by dissolving accurately weighted

amounts in 96% ethanol (v/v) and stored at -20 °C. 2C-B tablets were generously provided by

the Institute of Criminalistics Prague, Czechia.

2.2. Instrumentation

1

NMR spectra were recorded on a Varian Gemini 300 (300 MHz for H; 75 MHz for ¹³C) or a

Bruker Avance III HD 500 MHz (¹H NMR 500 MHz, C NMR 125 MHz) spectrometers. High

13

resolution mass spectra were measured on a LTQ Orbitrap Velos (Thermo Scientific)

spectrometer using ESI ionization technique. Mass spectra of hapten-protein conjugates were

measured on a Bruker Autoflex Speed MALDI-TOF/TOF spectrometer. Infrared spectra were

measured on a FTIR Nicolet 6700 (Thermo-Nicolet) spectrometer coupled with ATR cell

GladiATR (Pike). Automated column chromatography was carried out using a CombiFlash Rf

200 apparatus (Teledyne ISCO), for reverse phase chromatography prepacked Redisep Rf

Gold C18 columns (packed with C18-reverse phase silica gel) were used. Microplate reader

uQuant BIO-TEK was from Inc. Winooski, USA. Linomat V (Camag AG, Switzerland) and a

strip cutter (Economic Cutter ZQ2000, Shanghai Kinbio Tech Co., China) were used for the

preparation of immunostrips.

2.3. Buffer solutions

The following buffers were used in ELISA: coating buffer (0.01M carbonate/bicarbonate

buffer pH 9.6); assay buffer (PBS, 0.01M phosphate buffered saline, pH 7.4); washing buffer

(PBSTw, 0.05% Tween 20 to PBS (v/v)); substrate solution (9 mL 0.05M citrate/phosphate

buffer pH 5.0, 1 mg of TMB, 1 mL of DMSO, and 2 µL of 30% H₂O₂(v/v)); stop solution

(2M sulphuric acid).

The following buffers were used in immunochromatographic test (LFIA): coating buffer

(0.01M carbonate/bicarbonate buffer pH 9.6); assay buffer (0.1M borate buffer pH 8.8

containing Surfactant 10G (5% v/v), and PEG (2% w/v)); conjugate pad buffer (0.2M borate

buffer pH 8.8 containing BSA (1% w/v); sucrose (1% w/v) and Tween 20 (1% v/v)).

2.4. Synthesis of haptens (2C-H derivatives)

In order to obtain antibodies targeted on small molecules such as 2C-B or other

hallucinogenic phenethylamines, a suitable hapten must be designed, synthesized and attached

to a carrier protein, which elicits an immune response in an immunized animal. The structure

of the haptens was derived from 2,5-dimethoxyphenethylamine, 2C-H. The synthesis of two

2C-H derivatives bearing the linker with carboxylic functional group in the position 4 of the

benzene ring was proposed and carried out (Fig. 1). Spectral data of intermediates and the

final products are provided in Tab. 1.

1,4-Dimethoxy-2-(2-nitroethenyl)benzene (2)

To a solution of 2,5-dimethoxybenzaldehyde (1) (20 g, 120 mmol) and ethylenediammonium

diacetate (EDDA) (2.2 g, 12 mmol) in isopropanol (100 mL) nitromethane (7.8 mL, 140

mmol) was added. The solution was left to stir at room temperature. Overnight the solution

turned a deep orange and after 24 hours the stirring was discontinued. The precipitate was

filtered off and washed with a small amount of isopropanol. Drying in vacuo afforded 23.1 g

(92 %) of the title compound 2 as an intensely orange powder.

2-(2,5-Dimethoxyphenyl)ethan-1-amine (3) (Cheng et al., 1984)

Lithium aluminium hydride (8.71 g, 230 mmol) was suspended in dry tetrahydrofuran (180

mL). The suspension was cooled to 0 °C in an ice bath and it was stirred for 30 minutes. Then

solution of 2 (10.67 g, 51 mmol) in dry tetrahydrofuran (70 mL) was added dropwise to the

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reaction mixture. After the addition was complete, the reaction mixture was heated to reflux

overnight. The reaction mixture was allowed to cool to room temperature, before 2M solution

of sodium hydride (20 mL) and water (25 mL) were added. The formed aluminium salts were

filtered off and washed with tetrahydrofuran. The filtrate was dried over potassium carbonate

and tetrahydrofuran was evaporated. The crude product was purified by vacuum distillation

(128-132 °C, 1-3 torr) to afford 5.07 g (55 %) of the title compound 3 as clear oil.

N-[2-(2,5-Dimethoxyphenyl)ethyl]acetamide (4)

To a solution of 3 (5.07 g, 28 mmol) in dichloromethane (13 mL) triethylamine (TEA) (4.68

mL, 34 mmol) was added and the reaction mixture was stirred for 30 minutes. Then the

reaction mixture was cooled to 0 °C in an ice bath and acetic anhydride (2.78 mL, 29 mmol)

was added dropwise. When there was no starting material in the mixture (confirmed by TLC),

methanol (1.25 mL) was added. The reaction mixture was stirred overnight at room

temperature. Then the organic phase was washed with water twice (2 x 25 mL) and it was

dried over magnesium sulphate. The solvent was evaporated, and the crude product was

purified by column chromatography (dichloromethane/methanol 10:1) to afford 5.30 g (85 %)

of the title compound 4 in the form of colorless crystals.

Ethyl 2-[4-(2-acetamidoethyl)-2,5-dimethoxyphenyl]-2-oxoacetate (5)

A solution of 4 (0.15 g, 0.67 mmol) in dry dichloromethane (10 ml) under argon atmosphere

was cooled to -30 °C and titanium tetrachloride (0.14 ml, 1.25 mmol) was added dropwise.

Then a solution of ethyl chlorooxoacetate (0.10 ml, 0.92 mmol) in dry dichloromethane was

added dropwise to the reaction mixture and it was stirred at -30 °C for 30 minutes. The

mixture was allowed to warm to room temperature and stirring was continued for additional

27 hours. Then the reaction mixture was poured over crushed ice. After vigorous stirring, the

layers were separated, and the aqueous phase was extracted with dichloromethane three times

(3 x 50 ml). The organic extracts were combined and washed with water (50 ml), 5%

hydrochloric acid (50 ml), saturated aqueous sodium hydrogen carbonate (50 ml) and brine

(50 ml). After drying over magnesium sulphate, the solvent was evaporated. Purification by

gradient flash chromatography (dichloromethane/methanol, gradient 0 → 10 % methanol)

afforded 0.11 g (49 %) of the title compound 5 in the form of yellow viscous liquid.

Ethyl 2-[4-(2-acetamidoethyl)-2,5-dimethoxyphenyl]acetate (6)

A solution of 5 (0.30 g, 0.93 mmol) in acetic acid (57 ml) and 70% perchloric acid (0.28 ml)

was hydrogenated over 10% palladium on carbon (0.19 g, 1.80 mmol) for 7 hours at 0.3 MPa.

Then the catalyst was removed by filtration over celite. Acetic acid was evaporated, and crude

product was diluted with water (90 ml) and extracted with dichloromethane three times (3 x

60 ml). Combined organic phases were extracted with water (60 ml), saturated aqueous

sodium hydrogen carbonate (60 ml) and water (60 ml). After drying organic phase over

magnesium sulphate, the solvent was evaporated and 0.25 g (87 %) of the title compound 6

was obtained in the form of pale yellow solid.

4-[4-(2-Acetamidoethyl)-2,5-dimethoxyphenyl]-4-oxobutanoic acid (7)

A solution of 4 (3.50 g, 15.70 mmol) in dry dichloromethane (100 ml) under argon

atmosphere was cooled to -30 °C and titanium tetrachloride (3.20 ml, 29.20 mmol) was added

dropwise. Then a solution of methyl 4-chloro-4-oxobutanoate (2.70 ml, 21.70 mmol) in dry

dichloromethane (8 ml) was added dropwise to the reaction mixture and it was stirred at -30

°C for 30 minutes. The mixture was allowed to warm to room temperature and stirring was

continued for additional 23 hours. Then the reaction mixture was poured over crushed ice

(300 g). After vigorous stirring, the layers were separated, and the aqueous phase was

extracted with dichloromethane three times (3 x 250 ml). The organic extracts were combined

and washed with water (250 ml), 5% hydrochloric acid (50 ml) and brine (50 ml). After

drying over magnesium sulphate, the solvent was evaporated. Recrystallization from ethanol

afforded 3.16 g (62 %) of title compound 7 in the form of pale yellow solid.

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Methyl 4-[4-(2-acetamidoethyl)-2,5-dimethoxyphenyl]-4-oxobutanoate (8)

Sodium hydride in the form of 60% dispersion in mineral oil (0.03 g, 1.17 mmol) was added

to a solution of 7 (0.20 g, 0.60 mmol) in N,N-dimethylformamide (10 ml) and the reaction

mixture was stirred for 1 hour at room temperature. Then the reaction temperature was

lowered to 0 °C and iodomethane (0.10 ml, 1.55 mmol) was added dropwise. The reaction

mixture was heated to 60 °C and stirred overnight. Then it was diluted with water (20 ml) and

extracted with ethyl acetate three times (3 x 20 ml). Combined organic extracts were washed

with brine (30 ml) and dried over magnesium sulphate. Evaporation of the solvents afforded

0.19 g (94 %) of title compound 8 in the form of pale yellow solid.

Methyl 4-[4-(2-acetamidoethyl)-2,5-dimethoxyphenyl]butanoate (9)

A solution of 8 (0.12 g, 0.36 mmol) in acetic acid (24 ml) and 70% perchloric acid (0.13 ml)

was hydrogenated over 10% palladium on carbon (0.08 g, 0.78 mmol) for 4 hours at 0.3 MPa.

Then the catalyst was removed by filtration over celite. Acetic acid was evaporated, and crude

product was diluted with water (40 ml) and extracted with dichloromethane three times (3 x

25 ml). Combined organic phases were extracted with water (25 ml), saturated aqueous

sodium hydrogen carbonate (25 ml) and water (25 ml). After drying organic phase over

magnesium sulphate, the solvent was evaporated and 0.10 g (83 %) of the title compound 9

was obtained in the form of pale yellow solid.

2-[4-(2-Aminoethyl)-2,5-dimethoxyphenyl]acetic acid hydrochloride (10) - derivative of 2C-H

I

Compound 6 (0.13 g, 0.42 mmol) was treated with 2M solution of sodium hydroxide (3 ml).

The reaction mixture was heated to 120 °C and stirred for 20 hours at this temperature. After

spontaneous cooling to room temperature, concentrated hydrochloric acid was added to adjust

the pH value to 1. Then the solvent was removed under reduced pressure. Crude product was

purified by reverse phase chromatography (water/methanol 19:1) to afford 0.05 g (43 %) of

final compound 10 in the form of pale yellow solid.

4-[4-(2-Aminoethyl)-2,5-dimethoxyphenyl]butanoic acid hydrochloride (11) - derivative of

2C-H II

Compound 9 (0.09 g, 0.29 mmol) was treated with 2M solution of sodium hydroxide (1 ml).

The reaction mixture was heated to 120 °C and stirred for 20 hours at this temperature. After

spontaneous cooling to room temperature, concentrated hydrochloric acid was added to adjust

the pH value to 1. Then the solvent was removed under reduced pressure. Crude product was

purified by reverse phase chromatography (water/methanol 19:1) to afford 0.03 g (30 %) of

final compound 11 in the form of white solid.

2.5. Preparation and characterization of hapten-protein conjugates

The synthesized haptens were conjugated to BSA to form immunogens. Conjugates were

prepared by activated ester method, the conjugation step being carried out in reversed micellar

solution (Yatsimirskaya et al., 1993), and characterized by MALDI-TOF/TOF spectrometry.

Derivatives of 2C-H (1 eq) were dissolved in DMF (100 mg mL^-1). DCC (1.3 eq) and NHS

(1.3 eq) were added to the micro test tube, both in the form of solution in DMF (100 mg mL^-

¹). The reaction mixture was left to stand at room temperature until the by-product N,N’-

dicyclohexylurea spontaneously crystalized from the solution. After the reaction was

complete (confirmed by TLC), the crystals were separated and the solution with an activated

ester was directly used in subsequent reaction.

A solution of BSA (0.013 eq) in bicarbonate buffer (13 µg mL^-1) was mixed with a 0.3M

solution of Aerosol-OT (AOT, bis(2-ethylhexyl)sulfosuccinate) in octane to form a reversed

micellar solution. The activated ester of the hapten was added to these solutions with stirring.

The reaction mixture was stirred vigorously at room temperature overnight. The modified

BSA was precipitated by addition of cooled acetone and the suspension was centrifuged (4

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°C; 30 min; 1 500 g). The supernatant was then removed, and the collected precipitate was

washed with cooled acetone one more time. The suspension was again centrifuged (4 °C; 20

min). After the removal of the supernatant, the solid product was air dried. No additional

purification step was carried out. The obtained product was only dissolved in water and

lyophilised to afford the conjugate in a form suitable for immunization of laboratory animals.

For MALDI-TOF/TOF analysis, each conjugate was dissolved in distilled water (conc. 1 mg

mL^-1). The resulting solution was mixed with a matrix solution containing sinapinic acid

(conc. 10 mg mL^-1in 33% ACN/0,1% TFA) in a ratio of 1:2. The mixture (3 uL) was spotted

onto a MALDI plate and the solvents were left to vaporize. Mass spectra of hapten-protein

conjugates were measured on a Bruker Autoflex Speed spectrometer in linear mode with mass

range m/z 20 – 210 kDa. Obtained spectra were processed and evaluated in mMass software

(Strohalm et al., 2010).

The hapten 10 (15.3 mg, 55.5 μmol) was converted into the corresponding activated ester by

the reaction with DCC (14.9 mg, 72.1 μmol) and NHS (8.3 mg, 72.1 μmol). Subsequent

conjugation of the activated ester with BSA (44.4 mg, 0.74 μmol) afforded the immunogen

(60.7 mg).

Conjugate 2C-HII-BSA

The hapten 11 (11.0 mg, 36.2 μmol) was converted into the corresponding activated ester by

the reaction with DCC (9.7 mg, 47.1 μmol) and NHS (5.4 mg, 47.1 μmol). Subsequent

conjugation of the activated ester with BSA (29.0 mg, 0.48 μmol) afforded the immunogen

(31.1 mg).

2.6. Polyclonal antibodies production and cross-reactivity study

The conjugates 2C-HI-BSA and 2C-HII-BSA were used for immunization and polyclonal

rabbit antibodies were obtained. Immunization was performed in the company MediTox s.r.o.,

Konárovice, Czechia. Antibodies production was performed according to the standard

procedure (Lapcik et al., 2004): The appropriate conjugate was dissolved in a sterile isotonic

saline and was emulsified with the same volume of complete Freud's adjuvants. Subsequently,

it was applied subcutaneously to three or four spots on the rabbit's back and legs.

Immunization was repeated four times in 4-week periods, one dose representing 0.3 mg of the

immunogen in 0.3 mL of the emulsion. The final serum harvesting was realised 10 days after

the last immunization by the cardiac puncture under complete anesthesia. The collected

antiserum was lyophilized and stored at -28 °C. 1 mg of lyophilized antibodies were dissolved

in 1 mL of the PBS and stored at -28 °C.

To verify the ability of the antibodies to react with similar epitopes on different new

psychoactive substances, the cross-reactivity (CR) tests were performed. The calibration

curves for each of the tested substances were constructed. The CR (%) was calculated as (IC₅₀

of 2C-B) / (IC₅₀of the tested substance) x 100.

2.7. ELISA development

The indirect competitive ELISA (ic-ELISA) was performed according to the standard

procedure (Fojtikova et al., 2018): Plates were coated at 4 °C overnight with 50 ng mL^-1of

the 2C-HII-BSA in the coating buffer. Afterward, the unbound conjugate was removed with

the washing buffer (4 times, 350 µL/well). 50 µl/well of standard or sample in assay buffer,

followed by 50 µl/well of Anti-2C-B (2.5 µg mL^-1in the PBS-0.1% BSA w/v), were added.

Standard curves were prepared by serial dilutions from 0 – 5000 ng mL^-1in assay buffer. 1^st

incubation was allowed to take place for 1 h at room temperature. After washing, a 1:10 000

dilution of GAR-Po in assay buffer was added. 2^ndincubation was allowed to take place for 1

h at room temperature. Plates were then washed and finally, peroxidase activity was revealed

by adding 100 µl/well of a freshly prepared substrate solution (incubation at room

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temperature for 10 min). The enzyme reaction was stopped by adding the stop solution

(50 µL/well) and the absorbance was measured at 450 nm.

Interpretation of results

Sigmoid calibration standard curves were obtained by plotting the mean values of absorbance

against the logarithm of 2C-B concentrations through a four-parameter logistic equation as we

described previously (Fojtikova et al., 2016). The limit of detection (LOD) was defined as the

concentration of an analyte corresponding to the maximum assay signal minus 3x standard

deviation (SD) in accordance with the calibration curve (the blank was calculated from 3

parallel determinations with the absence of an analyte). The IC₅₀corresponded to the

concentration of analyte giving 50% inhibition of the asymptotic maximum. The linear

working range corresponded to the analyte concentration causing the 20-80% inhibition of the

maximum assay signal.

2.8. LFIA development

Gold-labelled antibodies

The gold-labelled antibodies (GAR-Au) were prepared according to the procedure previously

described by our group (Fojtikova et al., 2018) with a slight modification: to a gold colloid

nanoparticles suspension (gold colloid nanoparticles 0.5 mg mL^-1of 5 mM borate buffer, pH

8.8) GAR (1 mg mL^-1in PBS) was added. The mixture was stirred gently at 37 °C for 2 h.

Afterward, the suspension was centrifuged (10 °C; 60 min; 6 000g). The sediment was

washed 3× in a 5mM borate buffer, pH 8.8, containing BSA (1% w/v) and NaN₃(0.02% w/v)

using centrifugation (10 °C; 15 min; 13 640g). The final sediment was resuspended in 0.1M

borate buffer, pH 8.8, containing BSA (1% w/v) and NaN₃(0.02% w/v). Prepared stock

suspension of the GAR-Au conjugate (200 µg mL^-1) was stored at 4 °C in the dark.

LFIA procedure

LFIA strip was prepared based on previously described procedure (Fojtikova et al., 2018) with

several changes. The nitrocellulose membrane HiFlow Plus HFC18002 was stuck to the inert

backing material. The membrane was coated with DAG (conc. 75 µg mL^-1in coating buffer;

-1

2 µL mm ) at the control line and with 2C-HII-BSA (conc. 75 µg mL^-1in coating buffer;

-1

2 µL mm ) at the test line using the Linomat V. The conjugate pad was dispensed with the

-1

mixture of Anti-2C-B (1 mg mL^-1; 1.5 µL mm ) and GAR-Au (200 µg mL^-1; 10 µL mm ).

Prepared strips were put into LFIA cassettes and were stored in sealed bags under dry

conditions at laboratory temperature until used.

The 2C-B standard solution (the concentration range 0 – 10 000 ng mL^-1in assay buffer) or

sample (tablet, human urine) was added into the well of the LFIA cassette (100 µL/strip).

Subsequently, it migrated along the nitrocellulose membrane by capillary action. Positive

samples were identified by a decrease or the absence of red color intensity in the test line,

negative samples formed a red zone of maximum intensity on the test line. To obtain visual

detection limit (VDL), the strips of concentration series were compared with negative control.

The intensity of test line had to be significantly lower than the negative control. For

quantification, the strips were scanned and then processed with TotalLab.

2.9. Preparation of samples

Tablet extract

Analyzed tablet (weight 100 mg) contains 20 mg of 2C-B and its ethanol extract was prepared

as follows: the tablet was crushed, and the powder was mixed and extracted in 2 mL of 96%

ethanol (v/v) (room temperature, 20 min). Before LFIA analysis, the suspension was allowed

to stand for 10 min and then the supernatant was diluted in the assay buffer. Before ELISA

analysis, the suspension of the tablet was centrifuged (10 000×g, 10 min., room temperature)

and the supernatant was appropriately diluted in PBS.

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Urine sample

Samples of human urine were obtained from four laboratory volunteers (two women – sample

A, B; two men – sample C, D). Each of the freshly collected urine samples was spiked with

2C-B in order to obtain the concentration 10 ng mL^-1. Before ELISA analysis, the

contaminated samples were immediately centrifuged (10 000×g, 10 min., room temperature)

and the supernatants were appropriately diluted in PBS.

2.10. UHPLC-MS/MS analysis

For the UHPLC-MS/MS analysis Thermo Fischer Scientific UltiMate 3000 UHPLC system

coupled with Sciex QTRAP 6500 mass spectrometer (Sciex, Framingham, MA, USA) were

used. Chromatographic separation was performed on Phenomenex Kinetex PFP column (2.1 x

50 mm; 1.7 µm). The mobile phases for gradient elution were 0.1% formic acid + 2 mM

ammonium formate in water (A) and methanol (B). The flow rate was 0.37 mL min^-1and

column temperature 30 °C. Gradient elution was carried out as follows: 0 min, 98:2 (A:B); 1

min, 98:2; 4.75 min, 2:98; 6.75 min, 2:98; 8 min, 98:2; 10 min, 98:2. Retention time of 2C-B

was 6.72 min.

The MS/MS apparatus was operating in positive mode. The applied parameters of

electrospray ion source were: temperature 450 °C, curtain gas (CUR) 20 psi, nebulizer gas

(GS1) 30 psi and heater gas (GS2) 30 psi; ion spray voltage was set to 5350 V. Multiple

reaction monitoring (MRM) mode was used for the detection. Three transitions of m/z were

used (collision energies [eV] are given in brackets): 260 → 228 (30); 213 (35) and 164 (28).

Analyst Software (Sciex) was used for data acquisition and MultiQuant Software (Sciex) was

applied for quantification of samples.

Ethanol extract from the analysed tablet was centrifuged (10 000×g, 10 min., 15 °C) before

UHPLC/MS/MS analysis and the supernatant was appropriately diluted in ethanol. A serial

dilution (a series of three simple dilutions with dilution factors 0.2, 0.01 and 0.01,

respectively) was used. The sample was measured 7 times.

For calibration analytical standard of 2C-B was dissolved in ethanol and the solution was

appropriately diluted. Thirteen samples with concentrations of 2C-B ranging between 0.1 and

1000 ng mL^-1were prepared and measured three times. Peak areas of the external standard (at

each concentration) were plotted against the corresponding standard concentrations using

Hill’s regression with 1/y weighting to generate regression equation.

3. Results and discussion

3.1. Hapten design and synthesis

All the hallucinogenic phenethylamines are small molecules and therefore cannot elicit an

immune response unless they are attached to a large carrier protein. Hapten design plays an

important role in the development of an immunoassay in such a case. The structure of every

hapten should be as closely related to the target analytes as possible and it must

simultaneously incorporate a linker, which serves to join the hapten to a protein. In our case,

all the targeted drugs share 2,5-dimethoxyphenethylamine backbone and they differ only in

substituent in the position 4. Thus, two haptens bearing linkers in the position 4 were initially

proposed. The linker itself must be simple, as it should not become an epitope, once the

immunogen is applied to laboratory animals.

The proposed synthetic sequence started with the Henry reaction of 2,5-

dimethoxybenzaldehyde (1) and nitromethane. The resulting nitrostyrene derivative 2 was

reduced by lithium aluminium hydride to 2,5-dimethoxyphenethylamine (3) (Cheng et al.,

1984) and subsequently its primary amino group was protected by acetylation. Two different

linkers were introduced into the molecule of N-protected 2C-H 4 by Friedel-Crafts acylation,

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titanium tetrachloride served as Lewis acid in these reactions. Subsequent palladium-

catalyzed hydrogenolysis of the carbonyl group in the benzylic position and hydrolysis of

both ester and amide functional groups under basic conditions yielded the required haptens 10

and 11. In the case of intermediate 7, hydrolysis of the ester functional group was observed

during Friedel-Crafts reaction work-up. Thus, one extra step – alkylation of carboxylic ion –

had to be added into the sequence.

Two haptens with different linkers were prepared using a six-step synthetic sequence from

commercially available 2,5-dimethoxybenzaldehyde (1) with moderate to high yields in each

step. The synthesized haptens and all the intermediates were characterized by NMR and ESI-

MS (Tab. 1).

3.2. Preparation of hapten-protein conjugates and polyclonal antibodies production

The synthesized haptens were coupled to BSA. The modified BSA was simply precipitated

from the reaction mixture by addition of cooled acetone and separated by centrifugation. No

additional purification step was necessary, as only protein and hapten-protein conjugates are

precipitated under these conditions (Yatsimirskaya et al., 1993). The conjugate formation was

confirmed by a MALDI-TOF/TOF analysis. The hapten density (= an average number of

hapten molecules attached to the carrier protein) was calculated based on the obtained spectra

as the mass difference between the conjugated and the native protein divided by the mass of

the corresponding hapten (Wengatz et al., 1992). The binding ratio of the haptens to the

carrier protein was determined to be approximately 18:1 and 20:1 for the conjugates 2C-HI-

BSA and 2C-HII-BSA, respectively. The average number of haptens bound to BSA was

considered sufficient to elicit a specific immune response in immunized animals (Hu et al.,

2012).

Polyclonal antibodies obtained from the antiserum are supposed to be specific for a whole

group of structurally related compounds rather than for an individual molecule. This is of high

importance, as they could cross-react with higher number of hallucinogenic phenethylamines

currently present on the illicit market and moreover they could be advantageously employed

for the detection of newly emerging hallucinogens with similar structure.

3.3. Optimisation of the ic-ELISA and cross-reactivity study

Checkerboard titrations were used to select the most suitable antibodies and coating

conjugates (2C-HI-BSA and 2C-HII-BSA). Based on this study, conjugate 2C-HII-BSA and

corresponding antibody (Anti-2C-B) were chosen for the development of indirect competitive

ELISA (ic-ELISA). To determine the concentration of 2C-HII-BSA required for

immobilization and the concentration of Anti-2C-B antibody for an effective competitive

binding reaction, calibration curves were assembled. The immunoreactant concentrations that

gave absorbance value (A_max) of 1-1.9 and the lowest IC₅₀were considered as a reference

optimum binding condition. To enhance the sensitivity of the ELISA, several other conditions

(time and temperature of incubations, the composition of dilution buffers) were evaluated.

The optimized conditions are listed in details in the section Buffer solutions and ELISA

development.

According to the optimal conditions of the proposed ELISA, 12-point calibration curves

(concentration range 0 – 5000 ng mL^-1in PBS) were assembled and analytical parameters

were evaluated. The LOD was determined at 6 ± 3 pg mL^-1, the IC₅₀value of 1.9 ± 0.5 ng mL^-

1

and the linear working range of 0.1 – 31.0 ng mL^-1. The precision of the ic-ELISA is

possible to describe using intra-assay and inter-assay coefficients of variation. Therefore three

standard samples with 2C-B concentrations in the range of 0.5 – 5 ng mL^-1in PBS were

assayed. For the intra-assay precision study, one run of analyses (n = 3) was performed with

each sample on one plate. For the inter-assay, one run of analyses was carried out with each

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sample on three non-consecutive days. The resulting percent coefficients of variation were

calculated in the range of 7.3 – 13.8 %, indicating the acceptable precision.

In addition, the specificity of the Anti-2C-B was determined under the optimized ic-ELISA.

Cross-reactivity values were determined for 12 phenethylamines and several other new

psychoactive substances (10 cathinones, 8 cannabinoids, 6 piperazines, 1 aminoindane, 1

tryptamine). Obtained data for phenethylamines are summarized in Tab. 2. Based on the CR

results obtained with our ELISA, it can be stated that the Anti-2C-B is specific for tested

phenethylamines of the 2C sub-family (CR values were in the range of 34 to 100%). Anti-2C-

B can also be used to detect the DO sub-family (CR values were > 2%). The advantage of the

developed ic-ELISA is the possibility of using it for the detection of phenethylamines of both

sub-families, as the Anti-2C-B antibody binds not only 2C family members, but also DO

series drugs (CR values are shown in Tab. 2). Immunoassay for phenethylamines of the 2C

and DO sub-families was presented in patent literature, but the authors achieved the broad

cross-reactivity only by combining two different and separately developed antibodies

(Benchikh et al., 2018).

3.4. Optimisation of LFIA

By using 2C-HII-BSA as the conjugate immobilized on the test line, Anti-2C-B as the

primary antibody,

and

GAR-Au as

a

label,

an indirect competitive-type

immunochromatographic assay to detect 2C-B was established. Seven types of nitrocellulose

membrane were tested (PRIMA 85; AE 98; AE 99; AE 100; HiFlow Plus HFB13504; HiFlow

Plus HFB135UB; HiFlow Plus HFC18002). The best parameters (flow time; sharpness of

tested lines; signal intensity; test sensitivity) were observed when membrane HiFlow Plus

HFC18002 was used. The suitable amounts of the immunoreagents were chosen during a

checkboard titration experiment. Several amounts of 2C-HII-BSA conjugate immobilized on

-1

the membrane (75-200 µg mL^-1in coating buffer; 0.1 µL mm ) against the different amounts

-1

of Anti-2C-B (1 mg mL^-1; 1.5-4.0 µL mm ) were investigated. The concentrations of reagents

were chosen to satisfy the following facts: good sensitivity, minimum reagent consumption,

and acceptable color intensity and sharpness of the test line. The composition of the assay

buffer was also evaluated. Phosphate buffer and borate buffer of various pH values were

tested. Moreover, additives such as BSA (0.1-2% w/v); PEG (0.1-2% v/v) and trehalose (0.1-

2% w/v) and their combinations, with and without surfactants Tween 20 (0.1-5% v/v) or

Surfactant 10G (0.1-5% v/v) were tested to further improve the LFIA sensitivity. The

complete specifications of the optimized conditions are presented in section Buffer solutions

and LFIA development.

The sensitivity of the LFIA was determined using 2C-B standard solutions in the

concentration range 0 – 1000 ng mL^-1created in the assay buffer (three repetitions of the same

test). The visual detection limit (VDL) of the method was defined here as the concentration of

the standard in which the test line becomes less intensive compared with the negative control.

The color intensity of the test line was evaluated by naked eye. By comparing the color

intensity of the test lines, the VDL was determined as 10 ng mL^-1of 2C-B in the assay buffer.

To obtain the quantitative results, color intensities of the test lines were converted to pixel

grey volumes. The mean signal values were fitted to a sigmoid standard curve and analytical

parameters were obtained (the LOD was 15 ± 7 ng mL^-1; the IC₅₀value of 27 ± 2 ng mL^-1).

The precision study for LFIA was performed similarly as for ic-ELISA. Three standard

samples with 2C-B concentrations in the range of 0.1 – 10 µg mL^-1in assay buffer were

assayed, followed intra- and inter-assay coefficients of variation were determined (n = 3). The

resulting percent coefficients of variation were calculated in the range of 11.2 –30.5 %. The

cross-reactivity study for LFIA was also performed with the same synthetic drug standards as

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for ic-ELISA and all 9 substances mentioned in Table 2 were confirmed as significant cross-

reactant.

3.5. Analysis of 2C-B tablet using immunomethods and the UHPLC-MS/MS

The sample of the 2C-B tablet was prepared using extraction with organic solvent ethanol. In

case of immunoassays, the solvent can negatively change the native structure of antibodies

and influence condition of their interaction with antigen. The effect of ethanol on ic-ELISA

performance was tested by preparing 2C-B standard solutions in assay buffer containing

different amounts of solvent. Calibration curves constructed with mentioned standard

solutions (concentration range 0 – 5000 ng mL^-1in PBS containing 5-50% ethanol (v/v)) were

compared with a calibration curve constructed with standard solutions diluted only in assay

buffer (concentration range 0 – 5000 ng mL^-1in PBS). Based on the results (see Tab. 3), 10%

ethanol content was recommended as the maximum content in ELISA, higher concentrations

of ethanol affected sensitivity and IC₅₀values of relevant calibration curves greatly increased.

Ethanol was included in developed LFIA to evaluate its tolerance from 5 to 50% (v/v) solvent

content in standard solutions (conc. 0 or 1 µg mL^-1of 2C-B). The tolerance was evaluated

primarily on the basis of line intensities and the time needed for the analysis. It was explored

that the maximum ethanol concentration that can still be used for the developed LFIA is 20%

(v/v). This concentration does not have a significant effect on the test and control lines or time

of analysis. At higher ethanol concentrations, the duration of testing was prolonged, and

intensity of lines was reduced.

Ethanol extract of the 2C-B tablet was assayed three times by the newly developed ELISA

and LFIA and the results were compared with those obtained using UHPLC-MS/MS. Both

ELISA and HPLC methods yielded comparable results. The concentration of 2C-B in the

tablet determined by the ELISA was 0.204 mg/mg (RSD 4.6%), for the UHPLC/MS/MS it

was 0.205 mg/mg (RSD ranging between 1.77-2.37% depending on the chosen MRM

transition). The presence of 2C-B in the extract was also satisfactorily confirmed by the LFIA.

The concentration of 2C-B determined by the LFIA was 0.261 mg/mg (RSD 23%). To obtain

the quantitative results from the LFIA test, color intensities of the test lines were converted to

pixel grey volumes. The agreement of the results demonstrates a good potential of the

proposed immunomethods to be used for routine analyses of 2C-B.

3.6. Analysis of urine

At first, the influence of the human urine on the immunoassays was tested. To test for

possible interference by urine components in ELISA, 2C-B standard curves (concentration

range 0 – 5000 ng mL^-1) obtained in the PBS and in the presence of a variable proportion of

urine (0; 10; 20; and 30-times diluted in the PBS) were compared. Undiluted urine samples

significantly affect the obtained signal as well as IC₅₀values. However (see Fig. 2), 10-fold

diluted urine samples (sample A, B, C or D) do not affect the linear working range of

calibration curve, implying urine samples could be directly applied to the immunoassay

without difficult clean-up procedure (only diluted). Thanks to dilution of the samples, the

ELISA parameters will not be significantly affected and are comparable with commercially

available immunoassay screening kits (Regester et al., 2015) or with chromatographic

methods (Vorce et al., 2004; Al-Saffar et al., 2013) published previously.

The accuracy of the developed ic-ELISA was evaluated through recovery study with human

urine samples (A-D) spiked with 2C-B at level 10 ng mL^-1. The recovered amount of 2C-B

was calculated as (a concentration in urine sample measured by ELISA) / (spiked

concentration) × 100 (%). The recovery study was performed in four replicates and the results

(see Tab. 4) were satisfactory, all calculated data were in the range of 93 to 118 % with RSD

7.2 – 11.4 %.

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The similar experiments were used for LFIA. Aliquots of variously diluted urine samples (9:

1; 4: 1; 7: 3; 3: 2; 1: 1 in deionized water) were assayed with and without the addition of

standard (1 µg mL^-1of 2C-B). Standard solutions containing 0 and 1 µg mL^-1of 2C-B were

also assayed in the absence of urine sample. The intensity of the test lines was evaluated by

the naked eye (data not shown). The lowest possible dilution of the urine sample that does not

affect the intensity of the test line was the urine: water ratio 3: 2 (60 µL urine sample: 40 µL

water per strip). To obtain the lowest detectable concentration of 2C-B in urine samples

(VDL), 2C-B concentration range 0 – 10000 ng mL^-1were created in the urine and then

diluted in ratio 3:2 in deionized water. As the figure 3 shows, 25 ng mL^-1of 2C-B was

considered to be a VDL. The results demonstrate that LFIA developed for 2C-B detection in

urine is less sensitive than developed ic-ELISA. However, it is still comparable in sensitivity

to the previously published methods (Regester et al., 2015; Al-Saffar et al., 2013).

4. Conclusions

Two haptens derived from the structurally simplest substituted phenethylamine 2C-H were

designed, successfully synthesized and subsequently attached to bovine serum albumin. The

polyclonal antibodies obtained from rabbits immunized with the resulting conjugate 2C-HII-

BSA served for the development of two immunomethods aimed for the detection of 2C-B and

related hallucinogenic phenethylamines in human urine samples or extracts from confiscated

tablets. The presented LFIA is primarily suitable for on-site and roadside monitoring as it is

user-friendly and can provide a visual evidence of 2C-B presence in urine within a few

minutes. For greater accuracy, analyte presence should be verified by more sophisticated

method. In this study, highly sensitive ELISA is proposed for toxicological quantitative

analyses of positive samples captured by the LFIA. The comparison of the ELISA with the

well-established UHPLC-MS-MS method shows excellent agreement of results, which

confirms good potential of the ELISA to be used for routine analyses of 2C-B and related

hallucinogenic phenethylamines.

Acknowledgements

This work was financially supported by the Ministry of the Interior of the Czech Republic

(grant no. VI20172020056).

Declaration of interest

All authors declare no conflict of interest.

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ACCEPTED MANUSCRIPT

Table 1 Spectraldata of synthesized compounds.

Compound Spectral data

¹H NMR (300 MHz, CDCl₃) δ: 3.8 (s, 3H, OCH₃), 3.9 (s, 3H, OCH₃), 6.90 (d, 1H, J = 9.0 Hz,

2

ArH); 6.97 (d, 1H, J = 2.7 Hz, ArH), 7.01 (dd, 1H, J = 9.0, 2.7 Hz, ArH), 7.85 (d, 1H, J = 13.5 Hz,

CH), 8.11 (d, 1H, J =13.5 Hz, CH)

¹³C NMR (75 MHz, CDCl₃) δ: 55.82, 55.96, 112.37, 116.29, 119.12, 119.43, 135.26, 138.44,

153.48, 153.92

The spectral data are in agreement with those reported in Noland et al., 1999.

¹H NMR (300 MHz, CDCl₃) δ: 1.25 (br s, 2H, NH₂), 2.73 (t, 2H, J = 7.0 Hz, CH₂CH₂NH₂), 2.91 (t,

2H, J = 7.0 Hz, CH₂NH₂), 3.75 (s, 3H, OCH₃), 3.77 (s, 3H, OCH₃), 6.79-6.68 (m, 3H, ArH)

¹³C NMR (75 MHz, CDCl₃) δ: 34.94, 42.22, 55.55, 55.77, 111.16, 111.20, 116.81, 129.34, 151.86,

153.29

3

4

The spectral data are in agreement with those reported in the literature (Cheng et al., 1984; Bailey et

al., 1983).

¹H NMR (300 MHz, CDCl₃) δ: 1.92 (s, 3H, COCH₃), 2.80 (t, 2H, J = 6.8 Hz, CH₂CH₂NH), 3.44-

3.50 (m, 2H, CH₂NH), 3.76 (s, 3H, OCH₃), 3.80 (s, 3H, OCH₃), 5.70 (br s, 1H, NH), 6.72-6.79 (m,

3H, ArH)

¹³C NMR (75 MHz, CDCl₃) δ: 23.25, 30.20, 39.91, 55.64, 55.85, 111.34, 111.84, 116.73, 128.57,

151.65, 153.55, 169.99

The spectral data are in agreement with those reported in Longlois et al., 1995.

¹H NMR (300 MHz, CDCl₃) δ: 1.39 (t, 3H, J = 7.2 Hz, OCH₂CH₃), 1.94 (s, 3H, COCH₃), 2.88 (t,

2H, J = 10.4 Hz , CH₂CH₂NH), 3.46-3.53 (m, 2H, CH₂NH), 3.81 (s, 3H, OCH₃), 3.84 (s, 3H,

OCH₃), 4.39 (q, 2H, J = 7.2 Hz, OCH₂CH₃), 5.65 (br s, 1H, NH), 6.82 (s, 1H, ArH), 7.34 (s, 1H,

ArH)

5

¹³C NMR (75 MHz, CDCl₃) δ: 14.10, 23.24, 30.87, 39.15, 55.86, 56.73, 61.77, 110.41, 115.13,

120.96, 137.66, 152.03, 155.09, 165.54, 170.13, 185.91

HRMS (ESI): m/z [M + H]⁺calculated for C₁₆H₂₁NO₆: 324.14416, found: 324.14447; m/z [M +

Na]⁺

calculated

for

C₁₆H₂₁NO₆:

346.12611,

found:

346.12654

IR: 694, 1024, 1129, 1198, 1218, 1264, 1293, 1370, 1413, 1474, 1497, 1561, 1607, 1636, 1658,

1734, 2873, 2931, 3085, 3252

¹H NMR (300 MHz, CDCl₃) δ: 1.26 (t, 3H, J = 7.3 Hz, OCH₂CH₃), 1.92 (s, 3H, COCH₃), 2.80 (t,

2H, J = 6.7 Hz , CH₂CH₂NH), 3.43-3.49 (m, 2H, CH₂NH), 3.59 (s, 2H, CH₂CO), 3.76 (s, 3H,

OCH₃), 3.79 (s, 3H, OCH₃), 4.17 (q, 2H, J = 7.3 Hz, OCH₂CH₃), 5.76 (br s, 1H, NH), 6.68 (s, 1H,

6

ArH),

6.74

(s,

1H,

ArH)

¹³C NMR (75 MHz, CDCl₃) δ: 14.16, 23.20, 30.09, 35.69, 39.93, 55.88, 56.14, 60.60, 113.65,

121.77, 127.01, 151.10, 151.44, 170.05, 171.82

HRMS (ESI): m/z[M + Na]⁺calculated for C₁₆H₂₃NO₅: 332.14684, found: 332.14708

IR: 1043, 1169, 1185, 1214, 1295, 1321, 1343, 1365, 1402, 1434, 1515, 1562, 1647, 1729, 2830,

2929, 3321

¹H NMR (300 MHz, CDCl₃) δ: 1.93 (s, 3H, COCH₃), 2.77 (t, 2H, J = 6.4 Hz, CH₂COOH), 2.82 (t,

2H, J = 6.7 Hz, CH₂CH₂NH), 3.34 (t, 2H, J = 6.4 Hz, CH₂CO), 3.46-3.53 (m, 2H, CH₂NH), 3.73 (s,

3H, OCH₃), 3.84 (s, 3H, OCH₃), 5.58 (br s, 1H, NH), 6.78 (s, 1H, ArH), 7.11 (s, 1H, ArH), 11.82

7

(s,

1H,

COOH)

¹³C NMR (75 MHz, CDCl₃) δ: 23.36, 27.77, 30.84, 33.19, 39.22, 52.06, 56.06, 109.25, 117.21,

120.35, 138.90, 150.25, 157.06, 170.17, 173.26, 202.91

HRMS (ESI): m/z [M + H]⁺calculated for C₁₆H₂₁NO₆: 324.14416, found: 324.14453; m/z [M +

Na]⁺

calculated

for

C₁₆H₂₁NO₆:

346.12611,

found:

346.12667;

m/z [M + K]⁺calculated for C₁₆H₂₁NO₆: 362.10005, found: 362.10015

IR: 424, 451, 484, 536, 599, 725, 757, 776, 838, 887, 930, 983, 1027, 1063, 1136, 1167, 1181,

1201, 1223, 1257, 1299, 1366, 1409, 1434, 1467, 1495, 1550, 1617, 1634, 1722, 2848, 2916, 2945,

3078, 3295

¹H NMR (300 MHz, CDCl₃) δ: 1.93 (s, 3H, COCH₃), 2.70 (t, 2H, J = 6.4 Hz, CH₂COOCH₃), 2.86

(t, 2H, J = 6.7 Hz, CH₂CH₂NH), 3.33 (t, 2H, J = 6.4 Hz, CH₂CO), 3.45-3.52 (m, 2H, CH₂NH), 3.70

(s, 3H, COOCH₃), 3.82 (s, 3H, OCH₃), 3.88 (s, 3H, OCH₃), 5.66 (br s, 1H, NH), 6.79 (s, 1H, ArH),

8

7.35

(s,

1H,

ArH)

¹³C NMR (75 MHz, CDCl₃) δ: 23.37, 28.62, 30.62, 38.98, 39.65, 51.79, 55.94, 56.20, 111.82,

114.69, 125.47, 134.44, 151.48, 153.87, 170.20, 173.85, 198.86

HRMS (ESI): m/z [M + H]⁺calculated for C₁₇H₂₃NO₆: 338.15981, found: 338.16000; m/z [M +

Na]⁺

calculated

for

C₁₇H₂₃NO₆:

360.14176,

found:

360.14203;

ACCEPTED MANUSCRIPT

Compound Spectral data

m/z [M + K]⁺calculated for C₁₇H₂₃NO₆: 376.11570, found: 376.11594

IR: 488, 580, 636, 806, 849, 883, 936, 987, 1039, 1101, 1138, 1156, 1215, 1264, 1292, 1331, 1360,

1405, 1497, 1543, 1610, 1640, 1714, 2833, 2856, 2940, 3011, 3394, 3471

¹H NMR (300 MHz, CDCl₃) δ: 1.87-1.96 (m, 2H, ArCH₂CH₂), 1.93 (s, 3H, COCH₃), 2.34 (t, 2H, J

= 7.6 Hz, CH₂COOCH₃), 2.62 (t, 2H, J = 7.6 Hz, ArCH₂), 2.79 (t, 2H, J = 6.6 Hz, CH₂CH₂NH),

3.42-3.47 (m, 2H, CH₂NH), 3.66 (s, 3H, COOCH₃), 3.76 (s, 3H, OCH₃), 3.79 (s, 3H, OCH₃), 5.74

(br s, 1H, NH), 6.64 (s, 1H, ArH), 6.67 (s, 1H, ArH)

9

¹³C NMR (75 MHz, CDCl₃) δ: 23.42, 25.32, 29.64, 30.16, 33.70, 40.37, 51.54, 56.11, 56.17, 113.29,

113.49,

125.78,

128.91,

151.26,

151.61,

170.18,

174,24

HRMS (ESI): m/z [M + H]⁺calculated for C₁₇H₂₅NO₅: 324.18055, found: 324.18015

IR: 1043, 1141, 1207, 1240, 1291, 1367, 1406, 1435, 1510, 1553, 1647, 1732, 2869, 2928, 3336

¹H NMR (300 MHz, CD₃OD) δ: 2.98 (t, 2H, J = 7.1 Hz, CH₂CH₂NH₂), 3.18 (t, 2H, J = 7.1 Hz,

CH₂NH₂), 3.62 (s, 2H, CH₂COOH), 3.82 (s, 3H, OCH₃), 3.85 (s, 3H, OCH₃), 6.85 (s, 1H, ArH),

6.92 (s, 1H, ArH)

Hapten

10

=

derivative

of 2C-H I

¹³C NMR (125 MHz, CD₃OD) δ: 30.66, 37.56, 41.80, 57.21, 57.44, 115.54, 116.27, 125.92, 125.94,

153.58, 154.05, 177.18

HRMS (ESI): m/z [M + H]⁺calculated for C₁₂H₁₇NO₄: 240.12303, found: 240.12864

IR: 1038, 1185, 1212, 1237, 1400, 1411, 1467, 1512, 1704, 2652, 2743, 2894

¹H NMR (300 MHz, CD₃OD) δ: 1.89 (quint, 2H, ArCH₂CH₂), 2.31 (t, 2H, J = 7.5 Hz, CH₂COOH),

2.67 (t, 2H, J = 7.5 Hz, ArCH₂), 2.97 (t, 2H, J = 7.5 Hz, CH₂CH₂NH₂), 3.16 (t, 2H, J = 7.5 Hz

CH₂NH₂), 3.82 (s, 3H, OCH₃), 3.84 (s, 3H, OCH₃), 6.83 (s, 1H, ArH), 6.84 (s, 1H, ArH)

¹³C NMR (125 MHz, CD₃OD) δ: 27.35, 30.69, 31.41, 35.25, 41.88, 57.18, 57.30, 115.26, 115.41,

124.80, 132.11, 153.60, 153.83, 178.45

Hapten

11

=

derivative

of 2C-H

II

HRMS (ESI): m/z [M + H]⁺calculated for C₁₄H₂₁NO₄: 268.15434, found: 268.15496

IR: 845, 1042, 1187, 1208, 1281, 1377, 1401, 1427, 1464, 1507, 1701, 2903

ACCEPTED MANUSCRIPT

Table 2 Cross-reactivity data for Anti-2C-B in developed ic-ELISA.

Compound

2C-B

Structure

CR (%)

100.0

2C-I

84.0

42.9

2C-E

2C-P

2C-D

DOB

39.2

34.4

12.7

DOC

DOI

5.3

2.0

mescaline

0.9

CRs were less than 0.1% for other tested compounds.

ACCEPTED MANUSCRIPT

Table 3 Influence of the solvent ethanol on the characteristics of ELISA curve.

parameter

IC₅₀^b(ng.mL^-1)

Solvent

Ethanol

Solvent content (%, v/v)

0

1

5

10

20

30

50

Amax^a

1.09

1.10

1.08

1.10

1.07

1.11

0.90

1.7

1.9

3.5

4.6

8.6

^amaximum absorbance values

^bIC₅₀= the concentration of analyte giving 50% inhibition of the asymptotic maximum.

ACCEPTED MANUSCRIPT

Table 4 Recovery study with human urine samples (A-D) spiked with 2C-B.

Urine sample

Mean value

(ng mL^-1)

ELISA Recovery

(%)

RSD

(%)

Spiked concentration (ng mL^-1)

A

B

C

D

10

11.9

12.7

9.3

118

127

93

9.2

11.4

8.6

7.2

10.1

100

Set of analysis n=4;

Recovery = (a concentration in urine sample measured by ELISA)/ (spiked concentration) × 100 (%)

ACCEPTED MANUSCRIPT

Figure captions

Figure 1 Synthetic route leading to the derivatives of 2C-H.

[Artwork]

Figure 2 Effect of human urine sample (10-fold diluted in the PBS) on ic-ELISA performance.

[Artwork]

• PBS (IC₅₀2,03 ng·mL^-1); ♦ urine sample A (IC₅₀3,03 ng·mL^-1); ▲ urine sample B (IC₅₀2,17 ng·mL^-1);

■ urine sample C (I₅₀2,59 ng·mL^-1); ─ urine sample D (IC₅₀3,02 ng·mL^-1)

Figure 3 2C-B concentration series in human urine (diluted in ratio 3:2 in deionized water).

[Artwork]

A: 0 ng·mL⁻¹; B: 0.05 ng·mL⁻¹; C: 0.5 ng·mL⁻¹; D: 1 ng·mL⁻¹; E: 5 ng·mL⁻¹; F: 10 ng·mL⁻¹; G: 25 ng·mL⁻¹

(visual detection limit); H: 50 ng·mL⁻¹; I: 100 ng·mL⁻¹; J: 200 ng·mL⁻¹; K: 250 ng·mL⁻¹; L: 500 ng·mL⁻¹;

M: 1000 ng.mL^-1; N: 2500 ng.mL^-1; O: 5000 ng.mL^-1; P: 10000ng.mL^-1.

CL = control line; TL = test line.

Figure 1

Figure 2

Figure 3

Article Doi

Two immunoassays for the detection of 2C-B and related hallucinogenic phenethylamines

DOI: 10.1016/j.vascn.2018.11.001

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Article abstract of DOI:10.1016/j.vascn.2018.11.001

Full text of DOI:10.1016/j.vascn.2018.11.001

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