Determination of dopamine, serotonin, biosynthesis precursors and metabolites in rat brain microdialysates by ultrasonic-assisted in situ derivatization–dispersive liquid–liquid microextraction coupled with UHPLC-MS/MS
Graphical abstract
Introduction
Dopamine (DA) and serotonin (5-HT) are crucial monoamine neurotransmitters (NTs) that control many physiological and cognitive processes in humans. Their impaired metabolism has been implicated in several neurological disorders, such as Parkinson's disease (PD) characterized by alterations of nigrostriatal dopaminergic neurotransmission [1]. The gold standard pharmacological treatment over the past 40 years for PD is represented by oral administration of the DA precursor levodopa (L-DOPA). However, long-term L-DOPA treatment often leads to uncontrollable abnormal involuntary movements (AIMs) termed L-DOPA induced dyskinesias (LID), which affects approximately 90% of its users after a decade [2]. Increasing evidence points to the serotonergic system as a key player in the induction and expression of LID in both parkinsonian patients and animal models [3], [4]. Analysis of these NTs along with their biosynthesis precursors (L-DOPA, and 5-hydroxytryptophan, 5-HTP) and metabolites (3, 4-dihydroxyphenylacetic acid, DOPAC, and 5-hydroxyindole-3-acetic acid, 5-HIAA) provides a reliable insight into the activity of nervous system [5], [6]. Therefore, a fast, accurate and sensitive analytical method for the simultaneous determination of DA, 5-HT and their biosynthesis precursors and metabolites (shown in Table 1) in the brain is of great value in studying the role of NTs in LID and PD related diseases. With the advantages of non-traumatic merits, easy operation, in vivo continuous monitoring, economical application and so on, in vivo microdialysis (MD) sampling technique has enjoyed wide applicability in quantification for NTs [7], [8].
Various analytical methods have been widely investigated for determining the levels of monoamine NTs and their metabolites, including high performance liquid chromatography (HPLC) and capillary electrophoresis (CE) coupled with various detection techniques including ultraviolet (UV) detection [9], fluorescence detection (FLD) [10], [11], electrochemical detection (ECD) [12], chemiluminescence (CL) [13], and mass spectrometry (MS) [14], [15], [16]. Nevertheless, simultaneous measurement of these NTs is a challenging task hampered by difficulties derived largely from their low abundance, diversity in structure and physicochemical property, matrix interference, and potential instability. However, these methods have their own limitations, for example, low sensitivity and selectivity for UV and CL, bad repeatability for ECD due to electrode degradation, interferences after derivatization for FLD, and the difficulty of simultaneously separating NTs that have similar electrophoretic behavior for ECD. Compared with UV, FLD, ECD and CL, MS detection provides better sensitivity and specificity because analytes are identified by both retention times and molecular masses. Ultra high performance liquid chromatography coupled with tandem mass spectrometry (UHPLC–MS/MS) in multiple reaction monitoring (MRM) mode has become a powerful analytical technique for the determination of multiple NTs in different biological samples matrices [17].
Due to the low concentration levels, low sample volumes and high polarity of NTs in microdialysates, derivatization step was often employed in LC-MS in order to improve separations and increase the selectivity and sensitivity by introducing a charged or proton acceptor moiety into the functional group [18], [19]. The advantages of integrating derivatization with LC-MS analysis included: (a) improvement of selectivity and separation, (b) enhancement of ionization efficiency, (c) improvement of structural elucidation, (d) decrease of endogenous interference. Therefore, several derivatization methods coupled to LC-MS/MS were developed and applied for NTs determination. Using commercial dansyl chloride (Dns-Cl) as derivatization reagent, Zhao and Cai developed LC-MS/MS methods for the determination of amino acid neurotransmitters (AANTs) and monoamine neurotransmitters (MANTs) in rat plasma and human urine, respectively [16], [20]. Ji et al. reported a diethylation labeling approach for the determination of four monoamine NTs in rat prefrontal cortex microdialysates [21]. Benzoyl chloride derivatization method was used for multiple NTs by LC-MS/MS for microdialysates monitoring by Song [7] and Kovac [14], respectively. Recently, Greco et al. developed and validated a sensitive method for three monoamine NTs using (5-N-succinimidoxy-5-oxopentyl) triphenylphosphonium bromide as derivatization reagent [22]. However, these derivatization reagents have more or less limitations in their applications, such as poor stability, low detection sensitivity, operational inconvenience, difficult synthesis, or serious interferences in chromatogram. In this work, a commercial reagent with potential MS sensitizing effect, Lissamine rhodamine B sulfonylchloride (LRSC), was employed for the first time as derivatization reagent for multiple NTs in LID rat brain microdialysates.
Since the complex matrices of biological samples, an efficient pretreatment technique is essential. Dispersive liquid–liquid microextraction (DLLME) was introduced by Rezaee in 2006 [23] as a consequence of the demands for rapid, economical and environmentally benign sample-pretreatment techniques. In the past decade, various modifications of primary DLLME have been reported, including low-density solvent-based DLLME, ionic liquid-based DLLME, DLLME based on solidification of floating organic droplet, surfactant-assisted DLLME, shaker-assisted DLLME, vortex-assisted DLLME, ultrasonic-assisted DLLME, microwave-assisted DLLME [24], [25], dual DLLME [26], [27], lower-toxicity solvent-based DLLME [28] and novel automated DLLME [29]. The in situ derivatization is gaining more interest as sample preparation technique [30], [31]. In recent years, in situ derivatization combined with DLLME have attracted much attention as its potentiality to simplify experimental procedures, decrease sample loss, increase method sensitivity, and reduce matrix effects [32], [33].
In this work, a new ultrasonic-assisted in situ derivatization-dispersive liquid–liquid microextraction (in situ UAD-DLLME) method has been developed for the simultaneous determination of monoamine NTs, their biosynthesis precursors and metabolites along the DA and 5-HT metabolic pathway (Fig. 1A) in rat brain microdialysates by UHPLC-MS/MS in MRM mode. A commercial reagent LRSC was employed as derivatization reagent. The parameters affecting in situ UAD-DLLME and UHPLC-MS/MS were systematically investigated. Under the optimal experimental conditions, the analytical performance of the proposed method was evaluated, and the proposed method was successfully applied for monitoring the dynamics of NTs concentrations in rat brain microdialysates of LID rats.
Section snippets
Reagents and materials
Standards containing DA, L-DOPA, norepinephrine (NE), epinephrine (E), DOPAC, L-Tryptophane (Trp), 5-HTP, 5-HT and 5-HIAA (listed in Table 1) were purchased from Sigma (St. Louis, MO, USA). The internal standards (ISs) isoproterenol hydrochloride (Ip) and 5-hydroxyindole-2-carboxylic acid (5-HICA) [34] were analytical grade and purchased from Sigma (St. Louis, MO, USA). In this work, Ip was used as ISs for DA, L-DOPA, NE, E and DOPAC as shown in Fig. 1A, while 5-HICA was used as ISs for Trp,
UHPLC–MS/MS
The chromatographic conditions optimization was primarily focused to find a stationary phase, which was able to retain the NTs derivatives using suitable mobile phases. Among several different columns tested (Agilent Eclipse Plus C18, Agilent SB C18, Acquity BEH C18, Acquity BEH Shield RP18), best performances in terms of retention and separation were achieved when employing Agilent SB C18 column (theoretical plate numbers approached 12,000), a full end-capped C18 column with a mobile phase
Conclusions
A simple, green, rapid, efficient and sensitive in situ UAD-DLLME procedure combined with UHPLC-MS/MS method has been developed and validated for the simultaneous determination of NTs, their biosynthesis precursors and metabolites associated with LID in rat brain microdialysates. Higher sensitivity and selectivity with good precision, accuracy and matrix effect results of the method made it widely applicable for the routine analysis of multiple NTs in various biological samples. The use of a
Conflicts of interest
The authors declare that they have no conflict of interest.
Acknowledgements
This work was supported by the National Natural Science Foundation of China (Grant numbers 81303179, 21405094, 21475074, 21475075, 81573574, 81530094); the Natural Science Foundation of Shandong Province (Grant numbers ZR2013BQ018, ZR2013BQ019); the Open Funds of the State Key Laboratory of Electroanalytical Chemistry (No. SKLEAC201506); and the Foundation of Qufu Normal University (Grant numbers BSQD2012019, BSQD2012023).
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