Determination of Synthetic Impurities and Metabolic Products of F-ADAM, a Positron Emission Tomography Imaging Agent for Serotonin Transporter (SERT) Using HPLC-Tandem Mass Spectrometry *

A high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) analytical method was developed to determine the identity of impurities resulting from the synthesis of N,N-dimethyl-2-(2-amino-4-ﬂuorophenylthio)benzyl-amine (F-ADAM), as well as its metabolic products by rat liver microsomes. 18 F-ADAM is an im-portant positive electron emission ligand commonly employed as a ra-dio-imaging agent for serotonin transporter (SERT) in the brain. F-ADAM and its derivatives were separated using HPLC on a C4-phenyl column with an ammonium formate aqueous buffer/acetonitrile programmed mobile phase. Synthetic contaminants and metabolic products were identified using fragmentation spectra obtained by tandem mass spectrometry. We show that F-ADAM is unstable in methanol, and propose the use of acetonitrile to generate optimal chromatogram. A Cl-substituted species was found to be the major impurity resulting from the F-ADAM synthetic process. The metabolic products of F-ADAM by rat liver microsomes were characterized by oxidization of the sulfur moiety to sulfoxide, demethylation of the dimethylamine moiety, and oxidative defluorination/deamination. These results elucidate the by-products of F-ADAM synthetic and metabolic processes, and provide di-rection for the application of this imaging agent to biosystems properly.


Introduction
Serotonin is a critical neurotransmitter in the central nervous system (CNS) which regulates numerous physiological processes including mood, cognition, learning, memory, sleeping, depression, appetite, and aggressive behavior [1] [2] [3]. Serotonin transporter (SERT) regulates serotonin levels by reuptaking serotonin from the synaptic cleft [4]. Abnormal operation of the serotonin receptor and/or SERT has been linked to various mental disorders [4] [5]. Thus, SERT is the primary target of selective serotonin reuptake inhibitors (SSRIs), a group of drugs widely prescribed for the treatment of neurologic and psychiatric disorders such as depression, anxiety, suicide, schizophrenia, eating disorders, and drug addiction [6] [7] [8]. For the study of neurologic and psychiatric disorders in the context of treatment with SSRIs, non-invasive imaging approaches such as positron emission tomography (PET), and single photon emission computed tomography (SPECT) are powerful tools to guide drug development studies and evaluate SERT functionality [7] [8] [9] [10]. Because these techniques rely on radiolabelled molecular probe, it is critical to develop SPECT or PET-compatible radiotracers with high affinity and specificity for SERT [9].
The radio-isotopically labelled fluoride derivative of ADAM, 4-18 F-ADAM, was introduced in 2003 by Shiue and co-workers [14], and a facile synthetic procedure using a fully automated one-pot two-step method [15]. A series of studies of application of 18 F-ADAM to validate the ligand as a SERT imaging agent [7], applied in monkey brain [3], Parkinsonian rat models [16], and for the evaluation of neural transplantation treatment outcomes in Parkinsonian rats [17]. 18 F-ADAM PET SERT imaging has also been applied to evaluate brain function recovery in rats with 3,4-methylenedioxy methamphetamine (MDMA) induced behavior disorders treated with resveratrol [10]. Additionally, this group synthesized and compared various 18 F-labelled positions on the ADAM parent molecule for SERT binding affinity, showing that 4-18 F-ADAM was the best candidate for optimized SERT PET imaging [18]. Therefore, 4-18 F-ADAM holds great promise for the evaluation for serotonin-linked disorders.
The metabolic products formed from xenobiotic species when introduced into biosystems are directly related to their safety and efficacy, and must be characterized to ensure minimal toxicity [19]. Furthermore, for radio-labelled imaging agents, the integrity of the molecular probe must be maintained to maximize its contrast and affinity for the target organ. In general, biotransformation of chemical species into smaller or more polar molecules for detoxification and elimination from the body is accomplished by the CYP enzyme family. This process may also result in higher toxicity or, if intentionally exploited, pharmacological efficacy as in the case of prodrugs.
Similarly, the identification of concomitant impurities within drug substances and their unstable deteriorative products is paramount to ensure drug quality and safety [20]. The determination of impurities may also inform improved synthesis, purification, or storage conditions.
In this study, the fragmentation pathway of 4-19 F-ADAM (non-radioactive version) was elucidated by electrospray ionization triple quadrupole tandem mass spectrometry (ESI QqQ MS/MS). Additionally, an HPLC analytical method was employed to quantify impurities, derivatives, and metabolites of F-ADAM. The identity of synthetic impurities and metabolites resulting from rat liver microsome exposure were determined by tandem mass spectrometry. These results provide a more complete picture regarding the preparation and application of 4-18 F-ADAM.

Materials and Reagents
Analytical-grade chemicals for LC-MS were used as received without further purification. Acetonitrile (HPLC and MS grade), ammonium formate, and dimethyl sulfoxide (DMSO) were all purchased from Merck (Darmstadt, Germany). Ultrapure water (total organic carbon < 5 ppb, resistivity ≥ 18.2 MΩ-cm at 25˚C) was obtained using a Smart DQ3 reverse osmosis reagent water system (Merck Millipore, Billerica, MA, USA) fitted with a 0.22 μm polyvinylidene fluoride (PVDF) filter. Rat liver microsomes, solutions of NADPH coenzymes A and B, were all purchased from BD Biosciences (Bedford, MA, USA) and stored at −70˚C.
The compound of interest, 4-19 F-ADAM, was prepared by Shiue's group ( Figure 1). The product was characterized by TLC, melting point, 1 H NMR, and elemental analysis [14], and stored at −20˚C prior to analysis by HPLC or MS.

Analytical Equipment
The levels of F-ADAM impurities and metabolites were determined using an HPLC system (Agilent 1100/1200 series, Palo Alto, CA, USA) with an online degasser, binary pump, thermostat autosampler (10˚C), temperature-controlled column oven, and diode array detector (DAD, detection wavelength at 240 nm). Data were acquired and processed using Agilent ChemStation 10.02 software.
The HPLC system was coupled with a MS/MS apparatus (4000 QTRAP, AB Sciex, Concord, ON, Canada), operated using Analyst 1.6.2 software. The mass spectrometer was equipped with an electrospray ionization (ESI) source and a triple quadrupole linear ion trap (QqQ LIT) mass detector. The mass analyser was operated in positive-ion detection mode.

Analytical Method
The HPLC analytical method for F-ADAM employed a butyl-phenyl modified silica gel column (length 10 cm, i.d. 3 mm, particle size 3 μm, Gold Phenyl, Thermo Fisher) with a guard column. The column temperature was set at 23˚C.
The mobile phase was delivered by a two-pump programmed gradient (Table   1): pump A: formic acid 1 mL, ammonium formate 0.31 g dissolved in 1 L DI water and degassed in ultrasonic bath for 20 min; pump B: acetonitrile 100%.
The flow rate was 0.5 mL/min and the chromatographic time was 11 min per injection. The detection wavelength was 240 nm. The sample volume was 5 μL.

Procedures Used for Biotransformation Studies of F-ADAM
To generate metabolites of F-ADAM using rat liver microsomes, F-ADAM was dissolved in DI water-DMSO (1:1 mixture) as 1000 ppm and processed the solution following the guidelines for using of liver microsome [21] [22]. After periodic incubation (5, 30, 60, 90, and 120 min), the reaction was stopped by adding iced acetonitrile (1:1 volume) and vortexing, and the solutions were centrifuged at 6000 rpm at 4˚C for 10 min. The supernatant was filtered through a 0.22 μm disk membrane (PVDF) into a labelled HPLC vial and stored at −20˚C.

Mass Analysis of F-ADAM and Its Fragmentation Ions
Prior to identification of impurities/derivatives, mass analysis parameters for

Development of an HPLC Analytical Method for F-ADAM
The chromatogram of F-ADAM dissolved in methanol revealed that the peak of F-ADAM diverged after overnight storage at 10˚C with retention time (t R ) at 4.61 min and 4.88 min, respectively and peak height around 45:55. When the solution was freshly prepared, the resulting peak exhibited a t R at 4.7 min. The mass spectrum of the solution stored overnight showed an m/z for the impurity at 289, and the fragmentation spectrum is shown in Figure 4. Based on these results, it is believed that F-ADAM is unstable in methanol due to a methoxyl substitution at the fluoride position. It results from strong electronegativity and good leaving character for fluoride, while the solvent possesses nucleophilic reactivity. Thus, we suggested that F-ADAM should be dissolved in acetonitrile (or aprotic solvents) for the next works.   HPLC of F-ADAM was carried out on a butyl-phenyl modified silica gel column based on the π-π interaction of the phenyl and benzyl rings contained in the F-ADAM structure with the phenyl-modified column. By comparing the HPLC separation efficiency with methanol or acetonitrile as the pump B solvent, it is clear from the chromatographic results that acetonitrile is a more suitable solvent than methanol. The peak of F-ADAM was broader with methanol compared to acetonitrile (theoretical plate number, N, 10010 vs. 18500). Based on the optimal chromatographic characteristics, the formic acid aqueous buffer/acetonitrile gradient mobile phase coupled with the C4-phenyl column was employed for the study of F-ADAM. A typical chromatogram for F-ADAM is shown in Figure 5 with t R at 5.4 min, a dynamic range between 5 and 375 ppm, a correlation coefficient (r) > 0.9999 with slope 34.3 area unit per ppm, and an estimated lower limit of quantification (LLOQ) of 1 ppm by UV (240 nm). The chromatographic purity of F-ADAM was 97.5%.

Identification of Impurities within Solutions of F-ADAM
Several impurities coexisted with F-ADAM post-synthesis. The most significant impurity displayed a t R at 6.01 min with an aboundance of 2.14%. The first order mass spectra MS 1 revealed the impurity at m/z 311 and 313 with an intensity of about 2:1. These data suggested the compound contains a Cl moiety. Collision  Figure 7 indicate that the impurity is a chloro-substituted ana-logue. However, the position of the Cl moiety on the aromatic carbon is unclear. Thus, the structure showed in Figure 7 is one of possibility. An analysis of the synthetic process suggests this impurity is a by-product of starting material, 2-Cl-5-F-nitrobenzene.

Identification of Metabolites of F-ADAM Generated by Rat Liver Microsomes
The liver is the major organ responsible for transformation and degeneration of xenobiotics in the body. The fate of pharmaceutical agents in the body including processes such as adsorption, distribution, metabolism, and elimination (ADME) relate to drug safety and function. Importantly, a given molecule's metabolic scheme is related to the distribution and elimination of the drug. For radio-imaging agents, metabolism of the molecular probe impacts imaging quality, contrast, and precision. Liver microsome, which is derived from the membranes    Figure 10 shows the fragmented ion mass spectra for m/z = 263, a loss of 14 compared to F-ADAM representing demethylation, -CH 3 from dimethylamine. Figure 11 shows the fragmented ion mass spectra for m/z = 264, which was similar to Figure 10 with an m/z parallel shift of 1 unit. This may represent a hydroxyl (OH) group replacing the amino group on the phenyl moiety. Fragmented ion mass spectra showed that phenol does not release a hydroxyl group from the parent ion as indicated by the lack of a 264 → 246 transition, but rather shows a 233 → 215 transition accompanied by molecular rearrangement. The tandem mass spectrum shown in Figure 12 for a metabolite with m/z = 319 was an acetylation product at the phenylamine moiety. The fragmented ion mass spectrum and proposed structure for m/z = 321 are shown in Figure 13.

Conclusion
In this study of F-ADAM, the identity of unstable derivatives, synthetic impurities, and metabolites by hepatic enzymes were determined using electrospray ionization triple quadrupole tandem mass spectrometry and summarized in Figure 15 and Figure 16, respectively. These data reveal the origin of impurities  resulting from the F-ADAM synthetic process, as well as derivatives from its metabolic scheme. The metabolic scheme shows that the biosystem transforms the F-ADAM by increasing the molecular polarity, defluorination, deamination, and blocking the electron pair donating atoms (N and S) to metalloprotein.       Therefore, these metabolites do not bind to the SERT or PET traceable any more, which are decommissioned and eliminated from the body through urine. These findings may facilitate the optimization of the synthetic process and the proper use of F-ADAM as a PET radio-imaging agent for assessment of SERT function.