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 ) . 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 [ 68 ] .
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 .
nevertheless , tandem mass spectrometry ( ms / ms ) can provide high specificity due to additional structure information and high sensitivity .
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 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 . 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   c18 ( 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 .
the ( 6r)-5,6,7,8-tetrahydrobiopterin dihydrochloride , dopamine hydrochloride , serotonin hydrochloride , ( )-norepinephrine , ( )-epinephrine , d - glutamic acid , and -aminobutyric acid were purchased from sigma - aldrich corporation ( st . louis , mo , usa ) .
internal standards ( is ) with isotope labeling were 2-(3,4-dihydorxyphenyl ) ethyl-1,1,2,2-d4-amine hcl ( dopamine - d4 , 98% at % d ) ; serotonin-  , ,,-d4 creatinine sulfate complex ( serotonin - d4 , 98% at % d ) ; ( )-norepinephrine-2,5,6,,,-d6 hcl ( norepinepherine - d6 , 98% at % d ) ; ( )-epinephrine - d3 ( n - methyl - d3 ) ( epinephrine - d3 , 98% at % d ) ; l - glutamic-2,3,3,4,4-d5 acid ( glutamate - d5 , 98% at % d ) ; 4-aminobutyric-2,2,3,3,4,4-d6 acid ( -aminobutyric acid - d6 , 98% at % d ) .
epsilon - acetamidocaproic acid ( aaca ) was donated by kuhnil pharmaceuticals ( seoul , korea ) .
water was purified with a milli - q water purification system ( millipore , bedford , ma , usa ) .
full - scan positive mass spectra of bh4 and the is ( aaca ) reveal the protonated molecules , [ m + h ] , of m / z 242.1 and 174 , respectively
. the mass - to - charge ratios of fragments of bh4 after fragmentation were 166 , 107 , and 149 and fragments of is were 114 , 156 , and 79 .
the most abundant ion in the product ion spectra was 114 for is ( figure 1(a ) ) and 166 for bh4 ( figure 1(b ) ) .
in parallel , full - scan positive mass spectra of bh2 and the biopterin showed the protonated molecules , [ m + h ] , of m / z 240.0 and 238.0 , respectively .
the mass - to - charge ratios of fragments were 196.0 , 164.9 , and 168.0 in bh2 and of 177.9 , 193.9 , and 192.0 in biopterin .
the most abundant ion in the product ion spectra was 196.0 for bh2 ( figure 1(c ) ) and 177.9 for biopterin ( figure 1(d ) ) .
full - scan positive mass spectra of da and the is ( dopamine - d4 ) showed that m / z of protonated molecules [ m + h ] are 154.1 and 158.1 , respectively .
the mass - to - charge ratios of fragments of da after fragmentation were 137.0 , 90.9 , and 64.9 and of da - d4 141.0 , 95.0 , and 67.9 .
the most abundant ion in the product ion spectra was 137.0 for da ( figure 2(a ) ) and 141.0 for is ( figure 2(b ) ) .
but da mrm had 154.1 to 90.9 due to matrix effects , which increased the mass - to - charge ratio level .
full - scan positive mass spectra of 5-ht and the iss ( serotonin - d4 ) showed that the mass - to - charge ratios of protonated molecules [ m + h ] were 177.0 and 181.0 , respectively .
after fragmentation , fragments of 5-ht seen were m / z 160.0 , 114.9 , and 132.0 and fragments of 5-ht - d4  m / z were 164.0 , 118.0 , and 136.0 .
the most abundant ion in the product ion spectra was at 160.0 for 5-ht ( figure 2(c ) ) and at 164.0 for is ( figure 2(d ) ) .
full - scan positive mass spectra of ne and the is ( norepinephrine - d6 ) showed that the mass - to - charge ratios of protonated molecules [ m + h ] were 170.1 and 176.1 , respectively .
after fragmentation , fragments of ne were m / z 152.0 , 107.0 , and 76.9 and fragments of ne - d6  m / z were 158.0 , 111.0 , and 112.0 .
the most abundant ion in the product ion spectra was at 152.0 for ne ( figure 2(e ) ) and at 158.0 for is ( figure 2(f ) ) .
but m / z 170.1 to 107.0 and 176.1 to 111.0 was selected for ne and is ( ne - d6 ) mrm due to matrix effects , which increased the mass - to - charge ratio level .
full - scan positive mass spectra of ep and the is ( epinephrine - d3 ) showed that the mass - to - charge ratios of protonated molecules [ m + h ] were 184.1 and 187.1 , respectively .
the m / z of fragments of ep after fragmentation were 166.0 , 76.9 , and 107.0 and of ep - d3 169.0 , 76.9 , and 107.0 , respectively .
the most abundant ion in the product ion spectra was at 166.0 for ep ( figure 3(a ) ) and at 169.0 for is ( figure 3(b ) ) .
full - scan positive mass spectra of glu and the is ( glutamate - d5 ) showed that the mass - to - charge ratios of protonated molecules [ m + h ] were 148.0 and 153.0 , respectively .
the mass - to - charge ratios of fragments of glu after fragmentation were 129.0 , 83.9 , and 55.9 and of glu - d5 were 135.0 , 84.8 , and 88.0 , respectively .
the most abundant ion in the product ion spectra was at 129.0 for glutamate ( figure 3(c ) ) and at 135.0 for is ( figure 3(d ) ) .
but m / z 148.0 to 84.0 and m / z 153.0 to 88.0 for glutamate and is ( glutamate - d5 ) mrm were selected due to matrix effects , which increased the mass - to - charge ratio level .
full - scan positive mass spectra of gaba and the is ( gaba - d6 ) showed the protonated molecules , [ m + h ] , of m / z 104.0 and 110.1 , respectively .
the mass - to - charge ratios of fragments of gaba after fragmentation were 87.0 , 44.9 , and 85.0 and of gaba - d6 were 93.0 , 49.0 , and 91.9 , respectively .
the most abundant ion in the product ion spectra was at 87.0 for gaba ( figure 3(e ) ) and at 93.0 for is ( figure 3(f ) ) .
individual stock solution of each nt and isotope - labeled standard was prepared by accurate weighing of each compound ( 1  mg / ml methanol as the stock solution ) .
the solution of bh4 , da , 5-ht , ne , ep , glu , and gaba was prepared as a stock ( 1  mg / ml of each ) with pure acetonitrile and then diluted with acetonitrile ( 50% ) for each experiment .
standard solutions of bh4 , da , 5-ht , ne , ep , glu , and gaba for calibration curves were prepared by spiking the blank solution prepared to the appropriate amounts , but added volumes were less than 10% of total dw volume .
the final yielding concentrations for the standard curve were 10 , 20 , 50 , 100 , 200 , 500 , 1000 , 2000 , 5000 , and 10000  ng / g for bh4 and dopamine . in parallel ,
the final concentrations for the standard curve were 20 , 50 , 100 , 200 , 500 , 1000 , 2000 , 5000 , and 10000  ng / g for 5-ht , ne , and ep .
likewise , the final concentrations for the standard curve were 0.2 , 0.5 , 1 , 2 , 5 , 10 , 20 , 50 , 100 , and 200  g / g for glu and gaba .
the tolerance for reliable detection was 10  ng / g for bh4 and dopamine , 20  ng / g for 5-ht , ne and ep , and 0.2  g / g for glu and gaba .
the linear ranges and correlation coefficient of the calibration curve were summarized in table 1 .
icr mice ( male , body weight 2030  g , n = 24 ) , ( daehanbiolink inc . , chungju , south korea ) , were used .
the mice were kept under a controlled condition ( ambient temperature of 20 to 25c , 12-h light / dark cycle ) .
nih 's guidelines for animal research were followed for all animal procedures and were approved by institutional animal care and use committee ( iacuc ; dku-12 - 018 ) which adheres to the guidelines issued by the institution of laboratory of animal resources ( ilar ) .
the specific brain regions of mouse were quickly dissected on an ice bath   and , subsequently , isolated brain tissues were homogenized with acetonitrile ( 1  mg/10  l ) according to the internal standard ( aaca : 100  ng / ml ; dopamine - d4 , serotonin - d4 , norepinephrine - d6 , epinephrine - d3 , glutamate - d5 , and gaba - d6 :  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 4c .
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 .
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 hydrophilic interaction chromatography ( hilic ) sepax polar - imidazole ( 2.1  mm  100  mm , i.d . , 3  m particle size )
hplc column ( sepax technologies , delaware , usa ) to assay bh4 and dopamine , and luna 3   c18 ( 3.0  mm  150  mm , i.d . , 3  m particle size ) to assay serotonin , norepinepherine , epinephrine , glautamte , and gaba with a phenomenex c18 guard column ( 4  mm  2  mm , phenomenex ) .
a nanospace si-2 3004 column oven ( shiseido , japan ) was used online . 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 206c , 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 .
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 ) .
moreover , the peaks had a symmetric shape , and we confirmed the lc - ms / ms chromatogram of bh4 , aaca , da , and da - d4 ( 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 whole validation experiments followed the guideline of  fda ( us ) [ guidance for industry ; handling and retention of ba and be testing samples ] , may 2004 .
linear regression of the ratio of peak area of bh4 or nts to that of is was done with weighting of 1/x ( least - squares linear regression analysis , where x is the concentration of the analyte ) .
precision and accuracy were evaluated by three different concentrations of qc solutions : interday precision was evaluated for 5 replicates per a single concentration .
the value of accuracy was expressed as the mean of 25 replicates of determined concentration from 5 different analytical tests to the qc concentration ( table 2 ) .
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 t - test .
for simple sample preparation , protein precipitation was attempted using acetonitrile . to prevent sample degradation and oxidation , an ascorbic acid with 0.01% ( w / v )
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 ( c18 ) in chromatography analysis
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 .
the optimized electrospray ionization condition should be sensitive enough to detect bh4 , da , is , bh2 , and biopterin in positive ion detection mode .
the most abundant protonated ion peaks ( [ m + h ] ) in the q1 mass spectra of bh4 , da , is , bh2 , and biopterin were at 242.1 , 154.1 , 174.0 , 240.0 , and 238.0 , respectively ( figures 1 and 2 ) .
the product ions in q3 mass spectra and proposed fragmentation patterns were bh4 , which becomes at 2-amino-7,8-dihydropteridin-4(1h)-one of m / z 166.0 by losing propane-1,2-diol .
da becomes butane-1,2-diol of m / z 90.9 by losing ( e)-3-methylpent-3-en-1-amine ; is , ( e)-n - ethylidenepentan-1-amine of m / z 114.0 by losing both carboxyl and hydroxyl groups ; bh2 , 2-amino-7,8-dihydro-6-(hydroxymethyl)pteridin-4(1h)-one ) of m / z 196.0 by losing propan-2-ol ; biopterin , 2-amino-6-(hydroxymethyl)pteridin-4(1h)-one ) of m / z 196.0 by losing propan-2-ol . also , to confirm separation between bh4 and other biopterins in biological samples , experiments were previously conducted to quantify biopterin , bh2 , and bh4 in a mixed matrix .
the optimized electrospray ionization condition should be sensitive enough to detect bh4 , da , 5-ht , ne , ep , glu , gaba , and iss with positive ion detection mode .
the most abundant protonated ion peaks ( [ m + h ] ) in the q1 mass spectra of bh4 , da , 5-ht , ne , ep , glu , gaba , and iss are listed in table 3 .
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 .
previously , our lab reported bh4 and dopamine levels in rat brain region . to extend this method to mouse brain regions , we applied it the bh4 and nts in the mouse brain .
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 . 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 .
all analytes were subjected to hplc - ms / ms analysis , and their distinct mass - to - charge ( m / z ) ratios were determined .
there are representative lc - ms / ms chromatograms of bh4 , da , and iss ( aaca and dopamine - d6 ) 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 olfactory bulb ( ob ) , frontal cortex ( fc ) , hippocampus ( hp ) , striatum ( st ) , hypothalamus ( ht ) , pituitary gland ( pt ) , midbrain ( mb ) , cerebellum ( cb ) , and brainstem ( bs ) from the mice and subsequently confirmed that the quantity of bh4 and nts ( table 4 ) . eight different concentrations from 10 to 2000  ng / g of bh4 , from 10 to 5000  ng / g of da , from 20 to 10000  ng / g of 5-ht , ne , and ep , and from 0.2 to 200  g / g of glu , gaba is plotted against is for the standard curves .
the correlation coefficients ( r ) , lod , and loq of the standard curve are shown in table 1 .
the lc - ms / ms methodology was used to measure the levels of bh4 and nts in nine brain regions including ob , fc , hp , st , ht , pt , mb , cb , and bs from mice .
the newly - developed lc - ms / ms method was used to analyze the quantity of bh4 and nts in mice brain regions ( table 4 ) .
the endogenous levels of bh4 , da , 5-ht , ne , ep , glu , and gaba were successfully detected and measured in mice brain regions .
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 .
the other nts ( 5-ht , ne , ep , glu , and gaba ) were matched well with a luna 3   c18 column .
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 .
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 .
but the addition of antioxidant ( dte ) and/or acid ( hcl ) to the samples does not affect dramatically the stability of bh4 .
keeping the treated extracts at 4c 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 .
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  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 .
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 .
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 level of bh4 could be distinctly correlated with the level of da in the mouse brain tissue .
the 5-ht levels were at their highest in midbrain and brainstem , followed by hypothalamus , striatum , hippocampus , frontal cortex , occipital lobe , and cerebellum in a decreasing order .
the ne level was at its highest in hypothalamus and pituitary gland , followed by brainstem , midbrain , olfactory blub , cerebellum , striatum , and frontal cortex in a decreasing order .
the ep level was at its highest in hippocampus , followed by hypothalamus , brainstem , midbrain , olfactory blub , frontal cortex , and pituitary gland in a decreasing order .
however , there was no detectable ep in cerebellum ; the levels of ne and ep have similar order in mouse brain sections .
the glu level was at its highest in hippocampus , followed by frontal cortex , striatum , hypothalamus , cerebellum , midbrain , brainstem , olfactory blub , and pituitary gland in a decreasing order .
the gaba level was at its highest in hypothalamus , olfactory bulb , and hippocampus , followed by midbrain , frontal cortex , striatum , cerebellum , striatum , and brainstem in a decreasing order .
interestingly , the levels of glu and gaba were detected as microgram based units , but others were detected as nanogram based units .
these results suggested that the neurotransmitters in mouse brain were differentially released to do their function in brain sections .
however , a simple and rapid liquid chromatography tandem mass spectrometry ( lc - ms / ms ) method has been developed for the determination of bh4 , da , 5-ht , ne , ep , glu , and gaba in mouse brain ; the quantitative determination of endogenous neurotransmitters in brain regions by chromatographic coupled mass spectrometry presented here still has a limitation because of the typical lack of analyte - free matrix .
there is no analyte - free sample of the authentic matrix ; therefore , we have to use a surrogate matrix containing the authentic analyte .
a simple and rapid liquid chromatography tandem mass spectrometry ( lc - ms / ms ) method has been developed for the determination of bh4 , da , 5-ht , ne , ep , glu , and gaba in mouse brain using epsilon - acetamidocaproic acid ( aaca ) and isotopically labeled neurotransmitters as an internal standard . although it is clear that further studies are necessary to understand the physiological meaning of the different levels of bh4 and nts , this new method could be applied for tracking the changes of the endogenous bh4 and nts which are affected significantly by various stimuli or in neurodegenerative diseases [ 10 , 23 ] .
 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  m ) column by adding a mixture of 10  mm ammonium formate in acetonitrile / water ( 75  :  25 , v / v , 300  l / min ) for bh4 and da . to assay 5-ht , ne , ep , glu , and gaba ; a luna 3   c18 ( 3.0  mm  150  mm , i.d . , 3  m ) column was used by adding a mixture of 1% formic acid in acetonitrile / water ( 20  :  80 , v / v , 350  l / 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  g / 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 . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusions