MS spectra of their sodium adduct ions

Journal Pre-proof Structural elucidation of phenidate analogues via the ESI-MS/MS spectra of their sodium adduct ions Tamar Shamai Yamin (Conceptualization) (Methodology) (Writing review and editing) (Supervision), Hagit Prihed (Formal analysis) (Investigation), Moran Madmon (Formal analysis) (Investigation), Avital Shifrovitch (Formal analysis) (Investigation), Adva Baratz (Formal analysis) (Investigation), Avi Weissberg (Conceptualization) (Methodology) (Writing - review and editing) (Supervision)

PII:

S0379-0738(19)30456-6

DOI:

https://doi.org/10.1016/j.forsciint.2019.110044

Reference:

FSI 110044

To appear in:

Forensic Science International

Received Date:

5 September 2019

Revised Date:

24 October 2019

Accepted Date:

31 October 2019

Please cite this article as: Yamin TS, Prihed H, Madmon M, Shifrovitch A, Baratz A, Weissberg A, Structural elucidation of phenidate analogues via the ESI-MS/MS spectra of their sodium adduct ions, Forensic Science International (2019), doi: https://doi.org/10.1016/j.forsciint.2019.110044

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1 Structural elucidation of phenidate analogues via the ESI-MS/MS spectra of their sodium adduct ions

aAnalytical

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Authors Tamar Shamai Yamina, [email protected] Hagit Priheda, [email protected] Moran Madmona, [email protected] Avital Shifrovitcha, [email protected] Adva Baratza, [email protected] Chemistry Department, Israel Institute for Biological Research (IIBR), Ness Ziona,

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Israel.

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Corresponding author Avi Weissberga, [email protected], Tel: +972-8-9381570 , Fax: +972-8-9381688

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Graphical Abstract

Research highlights  EI-MS and ESI-MS/MS spectra of [M+H]+ of phenidate analogues are uninformative.  Different fragmentation patterns between [M+H]+ and [M+Na]+ were observed.  Structural information of phenidates was achieved only by ESI-MS/MS spectra of [M+Na]+.  Improved identification of novel phenidate analogues in water and urine was achieved.

2 Abstract The identification of phenidate new psychoactive substances (NPS) by implementing MS (Mass spectrometry) techniques is a challenging task. Phenidate analogues present information-poor mass spectra, both in GC-EI-MS and LC-ESI-MS/MS of the protonated molecules [M+H]+, with a high abundance fragment/product ion representing the secondary amine-containing residue. This lack of EI-MS and ESI-MS/MS information is attributed to the strong tendency of the

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amine residue to stabilize the positive charge and leads to unavoidable ambiguity in the

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identification process. Moreover, thermal decomposition of these compounds occurs in the

injection port and/or on the column under standard GC conditions. Herein, we demonstrate how

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structural information can be attained instantaneously through the LC-ESI-MS/MS

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fragmentation of the accompanied sodium adducts [M+Na]+. The sodium cation alters the charge distribution during ESI-MS/MS fragmentation, generating a major product ion corresponding to

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the Na+ adduction of the carbonyl group, providing new structural information of the main core of phenidate derivatives (alkylaryl acetate/acetic acid), enabling their reliable structural

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elucidation. This quick, simple and easy technique can be implemented to confirm the identity or identify various structurally related phenidate analogues in forensic toxicology and doping analysis without the need for sample handling.

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Keywords

LC-ESI-MS/MS, Phenidate analogues, structural elucidation, Sodium adduct, Forensic toxicology.

3 1. Introduction Methylphenidate is a central nervous system (CNS) stimulant and is an active ingredient in pharmaceutical products marketed under the names “Ritalin”, “Concerta” or “Rubifen”, which are used for the treatment of attention deficit hyperactivity disorder (ADHD) [1]. In the last decade, an array of phenidate analogues (a class of phenylethylamines), which are often more potent than methylphenidate, have been launched on the new psychoactive substances (NPS)

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market. Among them are 3,4-dichloromethylphenidate (3,4-DCMP) and ethylphenidate (EPH).

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In April 2015, the UK government imposed a temporary drug control order (TCDO) on all of these analogues [2]. The effects of NPS have rarely been studied and therefore pose a

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considerable and significant threat to the health of society. Forensic laboratories are often

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required to confirm the identity or to identify new psychoactive drugs without the availability of a reference standard or analytical data from the scientific literature. Therefore, an analytical

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technique for the identification of MPH-structurally related synthetic psychoactive drugs and their human metabolites, which may be used to confirm the consumption of the drug, is

analogues.

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necessary. This study aims to assist in the effort of reliable structural elucidation of phenidate

Several scientific articles have been published in recent years regarding the analytical characterization of phenidate analogues by nuclear magnetic resonance (NMR), infrared

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spectroscopy (IR), gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) [3–5]. The confirmation of such chemical identities by MS techniques turns out to be a challenging task because these analogues present information-poor mass spectra, both in GC-EI-MS and LC-ESIMS/MS analysis, with a fragment/product ion representing mainly or only the secondary amine-

4 containing residue. This lack of information is attributed to the strong tendency of the amine residue (piperidine) to stabilize the positive charge and leads to unavoidable ambiguity in the identification process. The availability of high-resolution accurate mass spectrometry (HRMS), which generally provides essential tools for the identification of unknowns of interest, including novel drugs and their metabolites [6], is also insufficient for the exclusive determination of the chemical structure of a molecule [7]. For structural elucidation, MS/MS spectra with diagnostic

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and informative product ions representing all parts of the molecules are essential [8].

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Protonation and alkali-metal cation adduction are the most important ionization processes in soft-ionization mass spectrometry. The formation of adducts in LC-MS depends mainly on

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their stability but is also influenced by ‘macroscopic’ factors [9,10]. The most common cation-

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forming adducts are sodium, potassium and ammonium in positive ionization mode [10]. Although the formation of sodium adducts in electrospray has been known for a long time, it has

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not been used extensively in practice.

Only a few studies have demonstrated that the adduct formation process can be applied

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successfully to solve different issues concerning the application of tandem mass spectrometry as a detector in liquid chromatography, such as sensitivity enhancement of oxygen-rich compounds [11–13], modification of the CID behavior of polymers (e.g., polylactides) [14] and in cases where the [M-H]- ion produces very small product ions or does not produce any product ions in

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the negative ion mode at all [15–17].

To the best of our knowledge, this is the first report on the use of adducts to solve a

problem related to the structural elucidation of small molecules in positive ion mode. We investigated the difference between the cleavage pattern of [M+H]+ and [M+Na]+, with the latter

5 producing more informative MS/MS spectra enabling instant structural elucidation of phenidate analogues without the need for chemical derivatization [18,19].

2. Experimental

2.1. Materials and reagents

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Methylphenidate (MPH) hydrochloride; 1.0 mg/mL in methanol, (±)-threo-3,4-

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dichloromethylphenidate (3,4-DCMP) hydrochloride; 1.0 mg/mL in methanol, ritalinic acid (RA) hydrochloride; 100 μg/mL in methanol, hydrochloric acid in ethanol (1.25 M), hydrochloric acid

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(37%), n-butanol and ammonium formate were obtained from Sigma-Aldrich (St. Louis, MO,

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USA). Water (LC-MS grade) and methanol (LC-MS grade) were obtained from Biolab Company (Jerusalem/Israel). Ethylphenidate and butylphenidate were prepared in-house by the microscale

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reaction of ritalinic acid and either ethanol or butanol in the presence of hydrochloric acid (HCl), according to the literature (Fischer esterification) [20]. A human urine matrix formulation

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(pooled anonymous donors) was purchased from ElSohly Laboratories, INC. (ELI).

2.2 Sample preparation

1 mL methanol was spiked with 1 μg of phenidate analogue prior to GC‐MS analysis.

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1 mL water was spiked with 100 ng to 10 μg of phenidate analogue prior to LC‐MS

analysis.

011 μL urine samples were spiked with 10 ng to 1 μg of phenidate analogues. After a

1:10 dilution with water, the mixture was analyzed by LC-MS.

6 2.2. Instruments and methods An HPLC-Q-Exactive Plus Orbitrap MS system was used. The analytes were separated using an Agilent 1290 high-performance LC system (Palo Alto, CA, USA) comprising a 1290 infinity binary pump with a jet weaver V35 mixer, a 1290 infinity autosampler and a 1290 infinity thermostatted column compartment (TCC). HPLC conditions: gradient elution was performed on a reverse-phase separation column

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(Gemini C18, 3.0 µm, 150 mm, 2.1 mm ID, Phenomenex, Switzerland) with a flow rate of 0.3

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mL/min. The column was maintained at 40℃. The gradient program (solvent A, water with 5% MeOH containing 1 mM ammonium formate; solvent B, MeOH containing 1 mM ammonium

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formate) was as follows: 0 to 10 min, a linear increase from 0% B to 95% B, a hold of 6 min at

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95% B, followed by a fast return to 100% A and a 4 min equilibration period. MS and MS/MS experiments were carried out with a Thermo Scientific Q-Exactive Plus

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Orbitrap MS (Thermo Fisher Scientific, Bremen, Germany) equipped with a heated-ESI (HESI) source operated in the positive ion mode.

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The Orbitrap HESI operating parameters were as follows: electrospray voltage, 1.25 kV; sheath gas flow rate, 45 (arbitrary units); auxiliary gas flow rate, 10 (arbitrary units); sweep gas flow rate, 2 (arbitrary units); aux gas heater temperature, 400 ℃; and capillary temperature, 275 ℃. The instrument was calibrated using a positive ESI calibration solution prepared according to

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the operating manual. All samples were analyzed using 2 alternating experiment types: full scan mode (m/z 60-500) at a resolving power of 70,000 and a 1x106 automatic gain control (AGC) target and a data-independent acquisition (DIA) experiment with an inclusion list at a resolving power of 35,000 and a 5x105 AGC target. The collision energy (CE) was set between 10 and 60 V.

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3. Results and discussion

3.1. GC-EI-MS analysis of phenidate analogues The GC-EI-MS spectra of phenidate analogues did not exhibit signals for the molecular ions and were dominated by the base peak at m/z 84. The thermal degradation of phenidate derivatives,

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e.g., methylphenidate, ethylphenidate, ethylnaphthalate and 3,4-DCMP, during GC analysis at

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temperatures above 200 °C has been reported by Flamm and Gal [21], Lee et al. [22] and Klare et al. [4], where the inconsistency of published mass spectra of several research groups has also

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been noted. This degradation occurs in the injection port and/or on the column under standard

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GC conditions. Small baseline peaks of methyl phenylacetate or ethyl phenylacetate partially coeluting with the methylphenidate and ethylphenidate, respectively, were observed. The GC-EI-

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MS spectrum of MPH is depicted in Fig. 1A. The mass spectrum of methylphenidate presents the common mass fragments m/z 150, 91 and 84. The mass fragment m/z 150 is the molecular ion of

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the thermal decomposition product, methyl phenylacetate. When coelution of methyl phenidate and methyl phenylacetate occurs, there is a contribution of the mass fragment m/z 91 as well as m/z 150 to the mass spectrum observed for the peak of methylphenidate (m/z 84). Moreover, the analogue 3,4-DCMP showed a strongly broadened major chromatographic peak, which can be

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attributed to the severe thermal degradation on the column via pyrolysis [4]. The GC-EI-MS spectra of 3,4-DCMP, which is illustrated in Fig. 1B, actually corresponds to the thermal degradation product, 3,4-dichlorophenyl methylacetate, which is consistent with a previous report [23]. Different GC-MS analyses present inconsistent mass spectra; therefore, nonstandard GC-MS methods were tested for their analysis efficacy [22], such as direct inlet mass

8 spectrometry (DI-MS) [23]. It is noteworthy that the hydrolysis products of phenidate analogues, which are usually their main metabolites, cannot be analyzed directly by GC-MS, and derivatization is necessary prior to their analysis. Therefore, LC-ESI-MS/MS is a superior alternative for the structural elucidation of both phenidate analogues and their major hydrolysis

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products.

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3.2. LC-ESI-MS/MS analysis of protonated phenidate analogues [M+H]+

MPH, EPH, BuPH, RA (their main metabolite), 3,4-DCMP and its hydrolysis product (3,4-

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DCRA), all bearing a piperidyl residue and presenting information-poor MS/MS spectra with a

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single, amine representative base peak (m/z 84.0808) with no information about the main core of

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the molecule (alkylaryl acetate/acetic acid), were chosen for analysis and evaluation (Fig. 2).

All six phenidate analogues, MPH (RT= 6.3 min), an aryl-modified MPH analogue (3,4-

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DCMP, RT= 8.4 min), their two hydroxycarbonyl analogues (RA, RT= 5.6 min and 3,4-DCRA, RT= 8.1 min), EPH (RT= 5.2 min) and BuPH (RT= 6.9 min), possess two moieties: an alkylaryl acetate/acetic acid and a secondary amine (piperidyl ring). The ESI-MS/MS spectra of all six phenidate analogues fragmented at a collision energy of 20 eV are shown in Fig. 3. In general,

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the secondary amine alone dominates the fragmentation pattern in ESI, regardless of the alkylaryl acetate/acetic acid moiety in the molecule. The Orbitrap-ESI-MS/MS spectra of these compounds reveal a single/major product ion at m/z 84.0808, which corresponds to the piperidyl ring in the molecule (2,3,4,5-tetrahydropyridine 1-ium-ion), generated by a loss of methyl phenylacetate (-150 Da) in MPH, ethyl phenylacetate (-164 Da) in EPH, butyl phenylacetate (-

9 192 Da) in BuPH, methyl dichlorophenylacetate (-218 Da) in 3,4-DCMP, phenylacetic acid (136 Da) in RA and a loss of dichlorophenyl acetic acid (-204 Da) in 3,4-DCRA. Ions representing the main core of the phenidates are hardly observed (<1%) in a broad range of collision-induced dissociation (CID) energies we utilized, making their identification based on MS/MS impractical.

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3.3. LC-ESI-MS/MS analysis of the sodiated phenidate analogues [M+Na]+

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To elucidate the structures of phenidate analogues, the dissociation processes of their cationic adducts were investigated. In the case of sodium, in addition to the formation of [M+Na]+, the

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formation of a dimer such as [2M+Na]+ was observed in addition to [M+K]+, though at a

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relatively low abundance in comparison to [M+Na]+. Therefore, we focused on the investigation of the predominant sodium adduct [M+Na]+. The Orbitrap-ESI-MS/MS spectra of the sodium

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10 eV are depicted in Fig. 4.

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adducts [M+Na]+ of MPH, EPH, BuPH, RA, 3,4-DCMP and 3,4-DCRA at a collision energy of

In contrast to the ESI-MS/MS spectra of the protonated phenidates [M+H]+, which present a single amine representative product ion (Scheme 1a), the ESI-MS/MS spectra of the sodium adducts [M+Na]+ reveal two competitive fragmentation pathways leading to the

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formation of two main product ions corresponding to the loss of the alkylaryl acetate/acetic acid (Scheme 1b1) and to the loss of 2,3,4,5-tetrahydropyridine (Scheme 1b2) for all six phenidate analogues.

10 The common product ion at m/z 106.0627 indicates the α-cleavage of the carbon-carbon bond adjacent to the piperidine and corresponds to the 2,3,4,5-tetrahydropyridine 1-ium-sodium ion. This ion is equivalent to the ion at m/z 84.0808 (2,3,4,5-tetrahydropyridine 1-ium-ion) observed in the ESI-MS/MS fragmentation of the protonated phenidate analogues [M+H]+. Furthermore, in the ESI-MS/MS spectra of the sodium adducts [M+Na]+, high abundance product ions, indicative of the alkylaryl acetate/acetic acid moiety, were also observed. These

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product ions were generated by the loss of 2,3,4,5-tetrahydropyridine from [M+Na]+ to produce

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sodiated adduct ions of phenyl methylacetate, phenyl ethylacetate, phenyl butylacetate, phenyl acetic acid, dichlorophenyl acetate and dichlorophenyl acetic acid in MPH, EPH, BuPH, RA,

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3,4-DCMP, and 3,4-DCRA, respectively. These two dissociation processes, which are observed

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for [M+Na]+, involve either the loss of 2,3,4,5-tetrahydropyridine or the loss of alkylaryl acetate/acetic acid and occur presumably via a concerted 6-member rearrangement reaction

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mechanism, which is common in the fragmentation of even-electron ions (ionic mechanism) in ESI [24,25]. Sugimura et al. [26] developed, based on a predominant trend with respect to adduct

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selectivity, the nitrogen-oxygen rule (NO rule) in methanol solutions for compounds containing nitrogen and oxygen atoms and demonstrated that compounds containing nitrogen but not oxygen atom(s) are preferably detected as [M+H]+ and compounds containing oxygen atom(s) are preferably detected as [M+Na]+. This is because oxygen bases have higher affinity to Na+

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than that of nitrogen bases due to the higher partial negative charge on oxygen atoms and competition for protonation in the case of nitrogen bases. Our investigated phenidate derivatives possess both nitrogen and oxygen atoms, and the observed predominant precursor ions were [M+H]+. [M+Na]+ adduct ions were also detected, however, at a concentration of two orders of magnitude lower.

11 The difference in fragmentation pattern between [M+H]+ and [M+Na]+ is attributed to the charge distribution alternation during ESI-MS/MS fragmentation. While the proton is preferably bound to the amine in [M+H]+, in [M+Na]+, the sodium cation possesses high affinity to the carbonyl group, withdrawing a large amount of electron density from the carbonyl oxygen and facilitating cleavage of the β-bond and migration of y-H towards the carbonyl oxygen. As a consequence, a dominant product ion (the sodiated adduct ion of alkylaryl acetate/acetic acid) is

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observed (Scheme 1b2). The formation of ions indicative of the alkylaryl acetate/acetic acid

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moiety enables their reliable structural elucidation. It is noteworthy that the sodium cation

induces a similar fragmentation pattern for all phenidate derivatives; however, the ratio between

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the abundances of the representative piperidine ion and alkylaryl acetate/acetic acid ion in the

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MS/MS spectra is varied. In four derivatives, an ion representative of the main phenidate core was dominant; however, in two other phenidate derivatives, 3,4-DCMP and 3,4-DCRA, the

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formation of the product ion corresponding to the main core was less favorable, and therefore, the two product ions were observed at similar intensities. This can be explained by the relative

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destabilization of the positive charge on the alkylaryl acetate/acetic acid moiety as a consequence of the two chloride substituents in comparison to the nonsubstituted phenyl moiety (MPH, EPH and BuPH). Although Na+ adduct ions were observed as the major adduct ions, some K+ adduct ions were also observed, though at relatively lower abundance, and they presented a similar

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fragmentation pattern to that of [M+Na]+.

3.4. Application in urine To demonstrate the applicability of the method in a real-world matrix, the ESI-MS/MS spectra of the sodiated phenidate analogues [M+Na]+ were also investigated in human urine samples.

12 Dilution with water (dilute-and-shoot) is the simplest and most rapid method of urine sample preparation for the measurement of drug metabolite concentrations by LC-ESI-MS/MS. Pharmacokinetic studies indicate that following 20 mg oral administration of fast or prolonged release MPH (the minimal effective dose is 20-80 mg daily), approximately 50% of the administered dose is excreted in the urine in the first 8 hours post-administration, primarily as RA, and its concentration is expected to be in the range between 4-14 µg/mL [27]. Therefore, a

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urine sample was spiked with RA and 3,4-DCRA at a concentration of 1 µg/mL (100 ng in 100

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µL urine sample), which is below the expected concentration level, diluted 1:10 with water and analyzed. The S/N ratios and peak shapes of the [M+Na]+ ions of both RA and 3,4-DCRA in

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diluted urine were compared to that in water and were found to be similar, with no observable

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matrix effect and no change in the intensity ratio between the [M+H]+ and [M+Na]+ ions, as depicted in Fig. 5 for RA. Blank samples prepared in the same procedure were analyzed in

retention times.

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between the samples. No carry over or co eluting interfering peaks were detected in the expected

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The developed method described here, enables qualitative determination (identification only) of phenidate derivatives, based on the ESI-MS/MS spectra of their [M+Na]+ ions, which provide improved mass spectral quality. Although for phenidate derivatives, [M+H]+ ions were observed as the major ion form, sodium ions originating from solvent and/or chemical

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impurities, glassware and the LC-MS system, lead to the formation of the accompanied sodium adducts [M+Na]+. These adducts were detected both in water and in human urine samples. The latter contain a sufficient concentration of Na+ ions even after dilution 1:10 with water, prior to analysis, which enables their detection at biologically relevant levels.

13 4. Conclusions Confirmation of the identity or identification of psychoactive drugs in forensic toxicology and doping analysis should be reliable with a high degree of certainty; therefore, detailed information about the molecular structure of the analyte is essential. Several recent official documents propose various new rules and strict assurance criteria. The criteria for complete identification or for drug identity confirmation are based upon the use of at least two different technologies (most

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often GC-MS and/or LC-MS), and a minimum of two diagnostic ions is mandatory in each MS

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analysis [28]. In the case of phenidates that are thermolabile compounds and their metabolites that are polar, GC-MS is considered the less advantageous technique, making LC-MS the

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method of choice; however, three diagnostic ions (1 precursor and 2 product ions) are not

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available using LC-ESI-MS/MS of the protonated molecules. Therefore, a second derivative should be prepared, or a different ionization and fragmentation technique should be used. Herein,

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we demonstrated that for phenidate analogues, complimentary data can be obtained through the ESI-MS/MS of the accompanied sodium adduct ions [M+Na]+. The ESI-MS/MS of [M+Na]+ has

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been proven as an essential tool for the structural elucidation of phenidate analogues and presented two main product ions indicative of the two parts of the molecule in contrast to a single amine representative product ion in the protonated molecular ion [M+H]+. This enables unambiguous identification of these compounds at sub µg/mL levels in water and in human

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urine, and as pharmacokinetic studies indicated that the concentration of phenidate metabolites such as RA is expected to be in the range of 4-14 µg/mL, this is below the expected concentration levels. To conclude, the sum of the total information obtained is called the “confirmation package”, which enables unambiguous identification of such phenidate analogues. The application of phenidate adduct ions to successfully solve problems concerning MS/MS

14 information is demonstrated for the first time. The developed analytical technique can be used for future structural determination of novel phenidate analogues and provide support for analytical data interpretation. Funding This research did not receive any specific grant from funding agencies in the public, commercial, or

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not-for-profit sectors

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Declaration of interest None

Credit author statement

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Tamar Shamai Yamin, Avi Weissberg: Conceptualization, Methodology, Writing - Review & Editing, Supervision. Hagit Prihed: Formal analysis, Investigation. Moran Madmon: Formal

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Formal analysis, Investigation.

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analysis, Investigation. Avital Shifrovitch: Formal analysis, Investigation. Adva Baratz:

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Fig. 1. GC-EI-MS spectra of 1 µg/mL solutions of MPH (A) and 3,4-DCMP (B); the latter

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corresponds to the thermal degradation product of 3,4-DCMP.

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Fig. 2. Structures of the phenidate analogues investigated in this study.

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Fig. 3. ESI-MS/MS spectra of the protonated phenidate analogues [M+H]+ at a collision energy of 20 eV. Formulae and ion structures were supported by Orbitrap‐MS2 exact mass measurements (<2 ppm error).

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Fig. 4. ESI-MS/MS spectra of the sodium adducts of the phenidate analogues [M+Na]+ fragmented at a collision energy of 10 eV. Formulae and ion structures were supported by Orbitrap‐MS2 exact mass measurements (<2 ppm error).

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Fig. 5. Full-MS chromatograms of protonated RA (A) and the sodium adduct of RA (B) in water

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(1 mL) spiked with 100 ng of RA and chromatograms of protonated RA (C) and the sodium adduct of RA (D) in a urine sample (100 µL) spiked with 100 ng of RA and diluted 1:10 with

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water. The ESI-MS/MS spectra of protonated RA (E) and the sodium adduct of RA (F) in the

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urine sample.

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Scheme 1. Plausible fragmentation patterns under ESI-MS/MS conditions for [M+H]+ (A) and for [M+Na]+ (B).