Quantification of Neurotransmitters in Mouse Brain Tissue by Using Liquid Chromatography Coupled Electrospray Tandem Mass Spectrometry

A simple and rapid liquid chromatography tandem mass spectrometry method has been developed for the determination of BH4, DA, 5-HT, NE, EP, Glu, and GABA in mouse brain using epsilon-acetamidocaproic acid and isotopically labeled neurotransmitters as internal standards. Proteins in the samples were precipitated by adding acetonitrile, and then the supernatants were separated by a Sepax Polar-Imidazole (2.1 mm × 100 mm, i.d., 3 μm) column by adding a mixture of 10 mM ammonium formate in acetonitrile/water (75 : 25, v/v, 300 μl/min) for BH4 and DA. To assay 5-HT, NE, EP, Glu, and GABA; a Luna 3 μ C18 (3.0 mm × 150 mm, i.d., 3 μm) column was used by adding a mixture of 1% formic acid in acetonitrile/water (20 : 80, v/v, 350 μl/min). The total chromatographic run time was 5.5 min. The method was validated for the analysis of samples. The calibration curve was linear between 10 and 2000 ng/g for BH4 (r2 = 0.995) , 10 and 5000 ng/g for DA (r2 = 0.997) , 20 and 10000 ng/g for 5-HT (r2 = 0.994) , NE (r2 = 0.993) , and EP (r2 = 0.993) , and 0.2 and 200 μg/g for Glu (r2 = 0.996) and GABA (r2 = 0.999) in the mouse brain tissues. As stated above, LC-MS/MS results were obtained and established to be a useful tool for the quantitative analysis of BH4, DA, 5-HT, NE, EP, Glu, and GABA in the experimental rodent brain.


Introduction
Neurotransmitters (NTs) are signaling molecules, which play pivotal roles in neuronal communications in the central nervous system [1,2]. It is reported that changes in NTs quantitation in several brain regions involve the development of many psychiatric diseases and neurodegenerative diseases [3,4]. Generally, neurotransmitters are classified into two categories based on their chemical styles: (i) the small molecules (dopamine (DA), serotonin (5-HT), norepinephrine (NE), epinephrine (EP), glutamate (Glu), -aminobutyric acid (GABA), histamine, and endocannabinoids and (ii) the neuropeptides (enkephalin, endorphin, and substance P) [5].
The quantitation of various NTs as a small molecule in the brain, especially the aromatic monoamines, should be measured by using high-pressure liquid chromatography (HPLC) separation coupled with amperometric electrochemical detection (ECD). This method has been applied in NTs analysis over the last three decades [6][7][8]. However, it is still rather difficult to determine different types of NTs simultaneously in one sample owing to the limited capability of accommodating changes in the mobile phase composition. Another difficulty of incorporating this method is that the analytes can only be identified by a stable retention time matching [9]. Nevertheless, tandem mass spectrometry (MS/MS) can provide high specificity due to additional structure information and high sensitivity [10]. Therefore, it has been commonly used for the quantification of NTs in the brain by coupling with both gas chromatography (GC) and liquid chromatography (LC) [5,11,12]. Owing to various efficiencies and time consumption of derivatization, a simplified sample preparation using liquid chromatography 2 Journal of Analytical Methods in Chemistry coupled with electrospray tandem mass spectrometry (ESI-MS/MS) is widely employed to quantify the NTs and their metabolites in the brains without derivatization [5,9,13]. The use of isotope labeled internal standards is vital to the enhanced method performance because the isotope ratio measurements provide a measure of quality control for each analyte by compensating for changes in analyte, retention time, recovery, degradation, and changes in detector responses caused by coeluting contaminants [9].
In this study, we developed a sensitive, simple, and simultaneous method to quantify the six major NTs such as DA, 5-HT, NE, EP, Glu, and GABA in mouse brains [14,15]. In addition, a tetrahydrobiopterin (BH4), a vital cofactor for the biosynthesis of the DA, 5-HT, and NE, was also measured in the same sample. To establish a novel method for the direct measurement of biologically active levels of BH4, DA, 5-HT, NE, EP, Glu, and GABA in the brain samples, the present study was performed using a high efficiency HILIC column for BH4 and DA, a Luna 3 C 18 (3.0 mm × 150 mm, i.d., 3 m) column for 5-HT, NE, EP, Glu, and GABA with reversed-phase HPLC separation and an ESI-MS/MS, which could minimize the sample interferences. At the same time, the multiple reactions monitoring (MRM) scan mode was sensitive enough to identify and quantify the BH4 and NTs in this new method.

Sample Preparation of Specific Brain
Regions. The specific brain regions of mouse were quickly dissected on an ice bath [16] and, subsequently, isolated brain tissues were homogenized with acetonitrile (1 mg/10 L) according to the internal standard (AACA: 100 ng/mL; dopamine-D 4 , serotonin-D 4 , norepinephrine-D 6 , epinephrine-D 3 , glutamate-D 5 , and GABA-D 6 : 1 g/mL). After a thorough homogenization, the BH4 and NTs (DA, 5-HT, NE, EP, Glu, and GABA) from brain tissues were extracted by sonication for 60s. The homogenates of brain tissue were centrifuged at 12,000 rpm for 10 min at 4 ∘ C. Supernatants were carefully transferred to 96-well plates and then injected onto the LC-MS/MS system by an  autosampler for subsequent analysis. For determination of BH4 and NTs in mouse brain tissues, d-water was used as blank matrix.

Apparatus and Chromatographic
Conditions. The liquid chromatographic system used was the Accela system (Thermo Fisher Scientific Inc., Waltham, MA, USA), equipped with a nanospace SI-2 3133 solvent delivery module as an autosampler (Shiseido Inc., Japan) and connected to Discovery Max (Thermo Fisher Scientific, Inc.) quadrupole tandem mass spectrometer coupled with electrospray ionization (ESI-MS/MS). System control and data analysis were performed using the Xcalibur software (Thermo Fisher Scientific, Inc.). Chromatographic separation was achieved using 6 Journal of Analytical Methods in Chemistry To assay BH4 and dopamine, the mobile phase consisted of 10 mM ammonium formate (pH 3) in an acetonitrile/water (75 : 25, v/v) mixture. The flow rate was 300 L/min and the injection volume was 5 L. To assay 5-HT, NE, EP, Glu, and GABA, the mobile phase consisted of an acetonitrile/water (20 : 80, v/v) mixture. The flow rate was run at 350 L/min and the injection volume was 5 L. The electrospray ionization (ESI) mass spectrometer was operated in the positive ion mode. The optimal condition was as follows: the ESI needle spray voltage was 4000 V, the sheath gas pressure 35 unit, the auxiliary gas pressure 20 unit, the capillary temperature 206 ∘ C, the collision gas (Ar) pressure 1.5 mTorr, the skimmer offset 5 V, and the chrome filter peak width 10 s. Scanning was performed in profile mode with the SIM width 0.700 FWHM, scan time 0.200 s, and scan width 0.5 Da.

BH4 and NTs Assay Method Developed Using LC-MS/MS. It was successful to qualify BH4 using Hydrophilic
Interaction Chromatography (HILIC) Sepax Polar-Imidazole (2.1 mm × 100 mm, i.d., 3 m particle size) HPLC column (Sepax Technologies, Delaware, USA). The BH4 and IS Peak was settled in a matrix-free region. Moreover, the peaks had a symmetric shape, and we confirmed the LC-MS/MS chromatogram of BH4, AACA, DA, and DA-D 4 (Figure 4). Following the same strategy, we analyzed successfully for 5-HT, NE, EP, Glu, and GABA by using Luna 3u C18 (3.0 mm × 150 mm, i.d., 3 m particle size). The peaks had a separated chromatogram and a symmetric shape ( Figure 5).  (Table 2).

Statistical Analysis.
All the values, tables, and figures given in the text are expressed as mean ± SD. Statistical differences between means were evaluated with two-tailed Student's -test. values less than 0.05 were taken to be statistically significant.

Sample Preparation and Liquid Chromatography.
For simple sample preparation, protein precipitation was attempted using acetonitrile. To prevent sample degradation and oxidation, an ascorbic acid with 0.01% (w/v) was also added and put in an ice bath. The peaks of BH4, dopamine, and IS were best when acetonitrile was used for protein precipitation and as an organic solvent of the mobile phase when using the HILIC column (Polar-Imidazole, 2.0 mm × 150 mm; i.d., 3 m) (Figure 4). Because BH4 and dopamine are easily dissolved in water, they are difficult to match the reverse column (C 18 ) in chromatography analysis. However, HILIC column can match well with the hydrophilic chemicals. This study used a Polar-Imidazole column in analyzing BH4 and dopamine. The other NTs (5-HT, NE, EP, Glu, and GABA) were matched with a C18 column (Luna 3 C18 (3.0 mm × 150 mm, i.d., 3 m particle size)) ( Figure 5), but the HILIC column could not separate peak of NTs clearly.  (Figures 1 and 2). There was no evidence of fragmentation and adduct formation.        Table 3. There was no evidence of fragmentation and adduct formation. The product ions and collision energy in Q3 mass spectra of BH4, DA, 5-HT, NE, EP, Glu, GABA, and ISs were listed in Table 3.

Sensitivity and Specificity of BH4 and NTs in the
Mouse Brain Tissue. Previously, our lab reported BH4 and dopamine levels in rat brain region [17]. To extend this method to mouse brain regions, we applied it the BH4 and NTs in the mouse brain. The standards for calibration were prepared by spiking them with DW. The peak areas of the spiked standard were constructed by subtracting the corresponding areas derived from the matrix. Meanwhile, calibration using internal standardization with deuterated analogues was performed. In biological specimen analysis, the isotope-labeled analogues of the targeted analyte are often recommended [9]. Due to their similar physicochemical properties, compared to deuterated analogues, the variability during sample preparation and ionization efficiency in the transfer of analytes from liquid to gas could be compensated for, and they could be differentiated ideally by their distinct mass-to-charge (m/z) ratios [18]. All analytes were subjected to HPLC-MS/MS analysis, and their distinct mass-to-charge (m/z) ratios were determined. The analytic parameters were listed in Table 1.
There are representative LC-MS/MS chromatograms of BH4, DA, and ISs (AACA and dopamine-D 6 ) in the DW matrix ( Figure 4). Also, there are 5-HT, NE, EP, Glu, GABA, and ISs LC-MS/MS chromatograms in the DW matrix ( Figure 5). We tested the newly developed method using

Discussion
The present study was undertaken in order to describe a sensitive and specific LC-MS/MS method for simultaneous detection of BH4, DA, 5-HT, NE, EP, Glu, and GABA from mouse brain tissue. The principal advantages of using LC-MS/MS method include a simple purification procedure and a simple chromatographic condition using the MRM scan mode. The use of a HILIC column overcame the limitations of separating hydrophilic materials. Therefore, HILIC column could separate BH4 and DA from matrix effect with an appropriate retention time [19]. The other NTs (5-HT, NE, EP, Glu, and GABA) were matched well with a Luna 3 C18 column. The quantitative and confirmatory assurance comes from coeluting isotopically labeled internal standards [15]. So, the current developed method should be very useful for brain tissue works of research, regarding the analysis of the alternation of the levels of BH4, DA, 5-HT, NE, EP, Glu, and GABA. This new method can enable measurement of BH4 and NTs rapidly and accurately in brain tissues. Previously, BH4 levels have been indirectly calculated by measuring the concentrations of biopterin in biological samples [18]. However the limitation of this indirect method is that it is unable to measure the exact BH4 levels owing to rapid oxidation and degradation. To avoid the problem, we tested several experimental conditions and found that a low temperature is a critical factor to prevent decomposition of BH4 in the brain tissues extract [17]. But the addition of antioxidant (DTE) and/or acid (HCl) to the samples does not affect dramatically the stability of BH4 [17]. Keeping the treated extracts at 4 ∘ C is necessary and enough to maintain BH4 stable for 4 hours, which is long enough to finish the analysis of the targets in samples. By using HILIC column, BH4 and DA were separated into single peaks. Under other methods, many unknown materials in the biological matrix interfered with the analysis of BH4 and DA in the biological samples [12]. But the use of a HILIC column could overcome the limitation to separating hydrophilic materials. So, HILIC column could separate successfully the BH4 and DA from matrix effect with an appropriate retention time (Figure 4). In addition, it could increase the sensitivity, selectivity, and accuracy of BH4 and DA in brain samples using MRM scan mode. Using Luna 3 C18 column (3.0 mm × 150 mm, i.d., 3 m particle size), 5-HT, NE, EP, Glu, and GABA were separated into single peaks. Also, the Luna 3 C18 column could separate NTs from matrix effect with an appropriate retention time, and the usage of MRM scan mode could increase the sensitivity, selectivity, and accuracy of NTs detection in brain samples ( Figure 5).
The levels of BH4 and NTs were measured in several brain sections by using the newly-developed experimental method ( Table 4). The BH4 is an essential cofactor for the aromatic acid hydroxylases, which are essential in the formation of NTs (DA, 5-HT, and NE), as well as for nitric oxide synthase (NOS), a vital enzyme for normal vascular and cardiac nitric oxide [20]. So, BH4 has been suggested to play a crucial role for many diseases. Therefore, it is necessary to reliably measure the biological concentration of BH4 for the evaluation of various diseases and for screening potential therapeutic candidates in neurological diseases [21]. These results showed that the BH4 level was at its highest in olfactory bulb, followed by cerebellum, frontal cortex, striatum, midbrain, pituitary gland, hypothalamus, brainstem, and hippocampus in a decreasing order. However, the DA level was at its highest in striatum, followed by hypothalamus, midbrain, pituitary gland, and olfactory bulb in a decreasing order. Interestingly, there were no detectable DA in hippocampus, frontal cortex, cerebellum, and brainstem. However, the lower limit of quantification in our method is 10 ng/g in samples. Therefore, even though there are some DA transmissions in these regions, the amount of DA in hippocampus, brain cortex, and brainstem could be below 10 ng/g. These data indicate that the