Dopamine hydrochloride (DA・HCl), (-)-norepinephrine (NE), serotonin hydrochloride (5-HT・HCl), L-ascorbic acid, sodium cyanoborohydride, formic acid, acetonitrile, methanol, ammonium acetate, paraformaldehyde, benzaldehyde and butyraldehyde were purchased from Sigma-Aldrich (Sanit Louis, MO, USA). Stable-isotopic labeling internal standards 2-(3,4-dihydroxyphenyl)ethyl-1,1,2,2-d₄-amine hydrochloride, (±)-norepinephrine-2,5,6,α,β,β-d₆ hydrochloride and serotonin-α,α,β,β,-d₄ creatinine sulfate complex (free base forms of the three internal standards were abbreviated as d₄-DA, d₆-NE and d₄-5-HT, respectively) were supplied by C/D/N Isotopes Inc. (Quebec, Canada). Ethanol absolute was supplied by Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Artificial cerebrospinal fluid (aCSF) was purchased from Harvard Apparatus (Holliston, MA, USA). Water was purified with an ELGA water purification system (ELGA, Bucks, UK).

Neurotransmitter standards DA・HCl, NE, and 5-HT・HCl were dissolved in 25 mM L-ascorbic acid in purified water: MeOH (v; v, 4:1) to obtain 1 mg/mL solutions of DA, NE, and 5-HT (concentrations were calculated in their free base forms), respectively. One mg/mL solutions were further multi-step diluted with 25 mM L-ascorbic acid in purified water: MeOH (v; v, 4:1) to generate stock solutions of DA, NE and 5-HT with the concentrations of 200 ng/mL, 20 ng/mL and 20 ng/mL, respectively. Calibration standards (CSs) were obtained by serial dilution of each stock solution with aCSF to the following concentrations: DA (25 pg/mL, 50 pg/mL, 100 pg/mL, 250 pg/mL, 500 pg/mL, 1000 pg/mL, 2500 pg/mL and 5000 pg/mL), NE (5 pg/mL, 10 pg/mL, 20 pg/mL, 50 pg/mL, 100 pg/mL, 200 pg/mL, 500 pg/mL and 1000 pg/mL), and 5-HT (2.5 pg/mL, 5 pg/mL, 10 pg/mL, 25 pg/mL, 50 pg/mL, 100 pg/mL, 250 pg/mL and 500 pg/mL). Quality control samples (QCs) were prepared by diluting stock solutions with aCSF to four different concentrations (DA: 25 pg/mL, 75 pg/mL, 400 pg/mL and 4000 pg/mL; NE: 5 pg/mL, 15 pg/mL, 80 pg/mL and 800 pg/mL; 5-HT: 2.5 pg/mL, 7.5 pg/mL, 40 pg/mL and 400 pg/mL), representing the limits of detection (LODs), low, medium and high concentrations of the calibration range. Internal standard (IS) solutions were prepared by individually dissolving salt forms of d₄-DA, d₆-NE, and d₄-5-HT in 25 mM L-ascorbic acid in purified water: MeOH (v;v, 4:1) to the concentration of 1 mg/mL (concentrations were calculated in their free base forms). Aliquot of each IS solution was further diluted with ethanol to obtain a mixture of d₄-DA, d₆-NE and d₄-5-HT with the concentrations of 5 ng/mL, 0.5 ng/mL and 0.5 ng/mL, respectively.

Ten μL of CSs, QCs, or microdialysate sample was transferred into a vial containing 10 μL of IS mixture, 25 μL of sodium cyanoborohydride solution (1.5 mg/mL in ethanol) and 25 μL of butyraldehyde: ethanol (v: v, 2:98). The mixture was vortexed well and incubated in 60˚C water bath for 80 min, and was further analyzed by ultra-high performance liquid chromatography?tandem mass spectrometry (UPLC-MS/MS).

In order to evaluate the linearity and sensitivity of the proposed method, CSs of DA (25 - 5000 pg/mL), NE (5 - 1000 pg/mL) and 5-HT (2.5 - 500 pg/mL) were prepared and derivatized according to the method above (Section 2.2 and Section 2.3). The calibration curves were constructed by plotting the peak-area ratio (analyte/IS) against the spiked concentration of analyte in CSs with duplicate measurements using linear regression. The limits of detection (LODs) were defined as the concentration with the signal-to-noise ratio greater than 3 (S/N > 3). The accuracy and precision were assessed by the recovery and coefficient of variation (CV, %) of QCs at four different concentrations. QCs at each concentration level were paralleled prepared in six replications on the day of analysis. The recovery was calculated by (calculated analyte concentration/nominal analyte concentration) × 100%.

Male Sprague-Dawley rat (body weight 280 - 300 g) was provided by SLAC National Accelerator laboratory (CA, USA). The room temperature of animal lab was 20˚C -25˚C and humidity was 40% - 70%. Animal was allowed free access to food and water. Animal was anesthetized with pentobarbital sodium (50 mg/kg, intraperitoneal). BASi^Ò BR-2 (2 mm membrane) brain microdialysis probes (Bregma polyacrylonitrile membrane, 30 KDa, 320 µm OD) (BASi corporate, IN, USA) was implanted with its tip at the upper limit of the striatum (coordinate in mm from Bregma: AP: +1.5; ML: +3.0; DV: −4.75). The probe was perfused with aCSF at 1.5 μL/min. After three days equilibration, microdialysis sampling was performed. Three consecutive microdialysate samples were collected at an interval of 20 min to determine the basal levels of DA, NE and 5-HT. Next, AMPH was intraperitoneally introduced at the concentration of 2 mg/kg. The concentration of AMPH, DA, NE and 5-HT were continuously monitored during a 220 min time course. Though 30 μL of microdialysate was collected every 20 min, only 10 μL was used for derivatization. The microdialysates were stored at −80˚C for future analysis. All the animal experiments were operated according to local ethical review and national legislation.

An aliquot of 3 µL brain microdialysate was protein precipitated with 60 µL acetonitrile containing 100 ng/mL labetalol as IS. The mixture was vortexed well for 1 min and centrifuged at 13,000 rpm for 15 min at 4˚C. Twenty-five µL of supernatant was transferred into a new tube and the solvents were blow-dried under N₂. The residue was then reconstituted with 50 µL purified water. The solution was vortexed well and centrifuged at 13,000 rpm for 15 min at 4˚C. Seven µL of supernatant was injected for UPLC-MS/MS analysis of AMPH.

Neurotransmitter analysis was carried out with a QTRAP^Ò 5500 mass spectrometer (Sciex, MA, USA) connected to an ACQUITY UPLC system (Waters, MA, USA) via an electrospray ionization (ESI) source. Separation protocol using an ACQUITY UPLC BEH C18 column (2.1 × 50 mm, 1.7 μm, Waters) was applied. The mobile phase gradient was set at 0.6 mL/min and 97% A for the first 0.2 min, and a linear gradient to 20% A at 1.2 min, and then to 2% A at 1.3 min, held 0.6 min, and returned to 97% A for 0.2 min to re-equilibrate the column, where A was acetonitrile:purified water (v: v, 5:95) containing 0.025% formic acid and 1 mM ammonium acetate and B was acetonitrile containing 0.025% formic acid and 1 mM ammonium acetate. The elution between 0.7 min and 1.0 min was flew into mass spectrometer, while the elution before 0.7 min and after 1.0 min were went to waste. Column temperature was 45˚C. The mass spectrometer was conducted in the positive mode with MRM transition. ESI settings were: source temperature was 600˚C, curtain gas was 40 psi, nebulizer gas pressure was 40 psi, auxiliary gas pressure was 60 psi and electrospray capillary voltage was 4.6 kV. MRM transition monitoring conditions were optimized for each compound and IS, and were summarized in Table 1. A 3.5 min separation protocol with a 3 μm-particle size, 2.1 × 100 mm ACE AQ column (Advanced Chromatography Technologies Ltd, Scotland) was applied for monitoring AMPH. The mobile phase gradient was set at 0.45 mL/min and 5% B for the first 0.3 min, and a linear gradient to 60% B at 2 min, and then to 95% B at 2.3 min, held 0.4 min, and returned to 5% B for 0.8 min to re-equilibrate the column, where A and B were consistent with the mobile phases utilized for derivatized MANTs. Column temperature was 45˚C. The mass spectrometer was conducted in the positive mode with MRM transition. ESI settings were: source temperature was 550˚C, curtain gas was 40 psi, nebulizer gas pressure was 50 psi and auxiliary gas pressure was 50 psi. MRM analysis parameters for AMPH were

described as follows: Q1 Mass (136.2 Da), Q3 Mass (119.2 Da), dwell time (50 msec), declustering potential (DP) (40 eV) and collision energy (CE) (12 eV) and retention time (1.49 min).

In this study, paraformaldehyde, benzaldehyde and butyraldehyde were investigated as derivatization reagents to react with DA, NE and 5-HT, respectively. Formaldehyde was reported as a common reagent to label amine groups [40] [41] . Dimethylated products were hard to retain on the RP column and eluted quite early together with other interference (data not shown), probably due to their strong polarity. Derivatization with excess benzaldehyde resulted in additional peaks right before the target compounds which would interfere their quantification (data not shown). Dibutylated DA, NE and 5-HT had good retention on the RP column, and the peak shape of these compounds were also satisfied. Therefore, we finally chose butyraldehyde as the derivatization reagent to perform reductive amination with DA, NE and 5-HT in this study (Figure 1).

The temperature and the duration of derivatization were optimized in order to obtain high conversion yield. Conversion yield was defined by Equation. (1), where PA represented the peak-area of target analyte at certain time point under certain reaction condition and PA_max was the highest peak-area could be obtained under the same condition.

Conversion yield ( % ) = P A P A max × 100 % (1)

The conversion yields for all three MANTs increased with time, and the rates were faster at higher temperatures (Figure 2). The highest conversion yields achieved after fourteen hours incubation under room temperature (Figure 2(a)) and reached maximum after 140 - 200 min at 37˚C (Figure 2(b)). DA, NE and 5-HT could achieve highest conversion yields within 80, 40 and 80 min at 60˚C, respectively (Figure 2(c)). Finally, incubation of MANTs with butyraldehyde at 60˚C for 80 min was chosen as the reaction condition for derivatization. The

products were stable at 10˚C after 26 hours of storage in the UPLC autosampler (data not shown). Different volumes of the derivatized MANTs (6 - 10 μL) were injected into UPLC/MS/MS system to investigate the effect of injection volume on the detection of MANTs. The results showed that the optimal peak shape could be obtained when the injection volume was 6.5 μL. Lower injection volume would reduce the intensity of the signals, while higher injection volume would result in split peaks (data not shown).

In general, reductive amination of DA, NE and 5-HT with butyraldehyde at 60˚C for 80 min provided a simple way to convent MANTs into corresponding dibutylated derivatives with high conversion yield. The current method was simple and did not require complex sample preparation steps, such as synthesis of the derivatization reagents or an UA-DLIME procedure. The optimal chromatographic peak shape of the derivatized products could be obtained with the injection volume of 6.5 μL.

The investigated linearity ranges of DA, NE and 5-HT were 25 - 5000, 5 - 1000 and 2.5 - 500 pg/mL, respectively. Good linearity correlations were obtained for the three MANTs with the coefficient of determination (R²) all above 0.99 (Table 2). The LODs (S/N > 3) for DA, NE and 5-HT were 25, 5 and 2.50 pg/mL, respectively (Table 2). Chromatograms of derivatized DA and their corresponding internal standards were presented in Figure 3. It was reported that the basal levels of MANTs were around tens to thousands of pictograms per milliliter in rat brain microdialysate [15] - [28] . Therefore, the above results suggested that current method had adequate sensitivity for simultaneous determination of DA, NE and 5-HT in rat brain microdialysate. The intra-day coefficient of variance (CV, %) of six batches of QCs were all below 13.4%, 12.0% and 8.5% for DA, NE and 5-HT, suggesting good precision and reproducibility of the proposed method. Recovery of all three compounds at different concentrations were within ± 15% of spiked amount. All validation data suggested that present method was reliable and reproducible, and could be applied to accurate quantification of DA, NE and 5-HT in the aCSF matrix.

The developed method was applied to simultaneously monitor DA, NE and

5-HT in SD rat brain microdialysates for a course of 220 min (Figure 4). Thirty μL of the microdialysate was collected at each time point, and only 10 μL was used for derivatization. Three consecutive microdialysates were collected before intraperitoneal injection of AMPH to monitor basal levels of MANTs. The basal concentration of DA (Figure 4(b)), NE (Figure 4(c)), and 5-HT (Figure 4(d)) were in the ranges of 143-159 pg/mL, 5.0-7.4 pg/mL and 14.7-25.0 pg/mL, respectively. Significant concentration increase was observed for all MANTs after AMPH administration. DA (Figure 4(b)), NE (Figure 4(c)) and 5-HT (Figure 4(d)) reached highest concentration after 40 min, 40 min, and 60 min injection of AMPH, respectively, and subsequently decreased with time.