Simultaneous Determination of the Neurotransmitters andFree Amino Acids in Rat Organs by LC-MS Analysis
Naoyuki Maeda1, 2※, , Michiko Sato2, Satoko Haeno2 and Hiroshi Yokota2＊1

Safety Research Institute for Chemical Compounds Co.,Ltd, Sapporo, Hokkaido 004-0839, Japan.
Laboratory of Veterinary Biochemistry, School of Veterinary Medicine, Rakuno Gakuen University,
Ebetsu, Hokkaido 069-8501, Japan.

2

Abbreviated Title: Determination of Free Amino Acids and Neurotransmitters※

Corresponding Author: Naoyuki Maeda, Ph.D.
Safety Research Institute for Chemical Compounds Co., Ltd
Sapporo, Hokkaido, Japan
Tel 81-11-885-5031
Fax 81-11-885-5313
Email: maeda-naoyuki@ka-anken.co.jp

Abstract
Free amino acids were kept at constant levels, and their alterations reflect the real state of energy metabolisms in the cells and living body. Neurotransmitters synthesized from certain amino acids fluctuated alongwith the current neuro-functional viability. Their content levels in each tissue give us substantial information about their state of health and disease in mammals. The determination method of free amino acids
without any derivative or labeling reactions and neurotransmitters in the various rat organs was developed
using a single column. The levels of free amino acids and neurotransmitters in the organs could be simultaneously and directly obtained within 80 min. Amino acids and neurotransmitters could be separated
within 20 min by using an “Intrada Amino Acid” column and identified by LC-TOF MS analysis, and they
were quantitatively analyzed by LC-MS/MS. Leucine and isoleucine could be completely separated bycolumn chromatography of the LC-MS analysis. The tissue specific localization of the amino acids andneurotransmitters obtained in this study can be physiologically explained. Additionally, the muscle specific branched-chain amino acids localization was found and the reasons were discussed.
Keywords:

Simultaneous assay, Free amino acid, Neurotransmitters, LC-MS analysis, Rat organs.

Introduction
Abnormal plasma and urinary concentrations of freeamino acids reflect the biosynthesis and catabolism ofproteins and energy metabolism in the cells of living
bodies (1). Amino acid metabolism, involving serine
and glycine, can provide the essential precursors forthe synthesis of protein, nucleic acids, and lipids thatare crucial to cancer metabolism (2, 3). Neurotransmitters and their metabolites are widely distributed in thebrain and peripheral body fluids of mammals (4, 5).

They are well known to play a significant role in the
nervous system, and they consist of amino acid neurotransmitters, such as glutamine and γ-aminobutyricacid; monoamine neurotransmitters, such as 5hydroxy-L-tryptophan,
5-hydroxytryptamine,
5hydroxyindoleacetic acid, L-dihydroxy-phenylalanine,
dopamine, norepinephrine, epinephrine and melatonin;
and others, such as acetylcholine. Neurotransmitter
imbalances caused by disturbances in the monoamine1metabolism and transporter have been linked to various neuronal abnormalities (6, 7). It would be possible
to recognize more real conditions of each cell if we
could determine the concentrations of the free aminoacids and neurotransmitters contained in the tissues
and organs. The monitoring of neurotransmitters andtheir metabolites is an essential tool for elucidating thenormal and pathological neuronal system activities.
Assays of the amino acids and neurotransmitters were
performed by HPLC, and they were detected by theultraviolet (UV) absorbance or derivative methods.
However, accurate and higher sensitivities could not
be obtained by UV methods. Additionally, suitablestabilities were not obtained by derivative methods (9- 12) because these methods were abandoned in response to their individual reactivities with derivatization reagents (13). Recently, using LC-MS analysis,
some investigators developed a method with lower
variability, good recovery and accuracy in the simultaneous determination of neurotransmitters (14 - 16).
Only neurotransmitters were measured with using
HPLC with ECD or GC/MS and LC-MS/MS previously reported (8, 15). However the comprehensive andsimultaneous data on the levels of free amino acids
and their metabolite neurotransmitters were significant
for understanding metabolite disorders and neuronaldiseases.
We developed an assay method with accurate andhighly sensitivity at the same levels as those describedabove for free amino acids and neurotransmitters contained in the various tissues of rat using LC-TOF MS
and LC-MS/MS, and their simultaneous actual levels
and new findings on muscular branched-chain aminoacids (BCAA; leucine, isoleucine and valine) wereobtained.
Materials and Methods
Chemicals and reagents - The following were purchased from Wako Pure Chemical Industries (Osaka,
JPN): amino acids - alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), cystine ((Cys) 2 ),glutamine (Gln), glutamic acid (Glu), glycine (Gly),
histidine (His), isoleucine (Ile), leucine (Leu), lysine(Lys), methionine (Met), phenylalanine (Phe), proline(Pro), serine (Ser), threonine (Thr), tryptophan (Trp),
tyrosine (Tyr), valine (Val), and 4-aminobutanoic acid
(GABA); stable isotopes - GABA-d 6 ; indoleamines Serotonin (5-HT), 5-hydroxytryptphan (5-HTP), 5Hydroxyindoleacetic Acid (5-HIAA), melatonin(MLT) and catecholamines - dopamine (DA), epinephrine(EP),norepinephrine(NE),L-3,4dihydroxyphenylalanine (L-DOPA) and neurotransmitter- acetylcholine (AC) were purchased from Sig-

ma Aldrich (St. Louis, MO). Stable isotopes, 5-HT-d 4
and DA-d 4 , were purchased from Taiyo Nippon Sanso(Tokyo, JPN). An acetonitrile and hexane for pesticide
residue analysis were purchased from Kanto Chemical(Tokyo, JPN). LC-MS grade acetonitrile, tetorahydrofulan, ammonium formate and formic acid werepurchased from Thermo Fisher scientific (San Jose,CA).
Preparation of rat organs - Male Sprague-Dawley(SD) rats (weight, 280 ± 20 g; age, 8 - 10 weeks) werefed, housed and allowed to adapt to their environmentsfor one week prior to the experiments. The investigation conformed to the Guide for the Care and Use ofLaboratory Animals published by the US National
Institutes of Health (NIH Publication No. 85 - 23, revised 1996). The blood and organs were prepared fromthe animals by exsanguination under isoflurane anesthesia. After dissection, the organ samples that were
excised post-mortem were weighed, immediately frozen and stored at -25 °C until use.
Preparation of samples for MS analysis - Male
Sprague-Dawley rat organs were obtained on ice immediately after death in the animal laboratory andtransported in liquid nitrogen to our laboratory; they
were frozen immediately at -80 °C until biochemical
analysis. The rat serum and brain tissue was homogenized in ice-cold 4:6 acetonitrile: water with 0.1 %formic acid (per gram of tissue by adding 4.0 mL ofsolution) after they were precisely weighed in the 50
mL centrifuge tube. Then, 1.0 mL of 100 μ mol /L of
DA, 5-HT and GABA of the internal standard wereadded to the tube and centrifuged at 8,000 rpm (10,160
×g, 7780II, Kubota Tokyo, JPN) for 20 min. Supernatant (1.0 mL) was pipetted into a 2.0 mL centrifuge
tube and 1 mL of hexane was added (saturated withacetonitrile); the solution was vortex-mixed for 10 sec,
centrifuged at 3,000 rpm (700 ×g, Kitman-18, TOMYTokyo, JPN) for 5 min, and finally centrifuged at
12,000 rpm (11,400 ×g) for 5 min using PVDF 0.22μm Ultrafree-MC (Millipore, Billerica, MA) filtrated.
The filtrated sample was injected into the LC-TOF MS
and LC-MS/MS analysis (Fig. 1).
Separation of the free amino acids and neurotransmitters - The separation was achieved using an
Intrada Amino Acid (100 × 3 mm, 3-μm particle size,
Imtakt Kyoto, JPN) at a 400 μL/min flow rate at 35 °C.
A 5 μL aliquot was used for the autosampler injection.
The positive ion mode scanning of a gradient mobile
phase, consisting of (A) acetonitrile: THF: 25 m mol/Lammonium formate: formic acid 10: 80: 10: 0.4 [v/v]
solution and (B) acetonitrile: 100 m mol/L ammonium
2

formate 20:80 solution, was used. For the gradientelution, (A)/(B) ratios were used from 100/0 hold at 1
min, to 83/17 and 0 / 100 for between 1 and 6.5 min
and between 6.5 and 10 min, respectively, which was
followed by an 8 min hold at 100 % (B) and a final
return to 100 % (A) within 4 min for a total 22 min
running time.
Quantification of free amino acids and neurotransmitters expressed in tissues - For the quantification of the free amino acids and neurotransmitters, aXevo TQD triple-stage quadrupole mass spectrometer
connected to an acuity H-class (Waters Midford, MA)
and an ESI ion source device was constructed (LCMS/MS). The MS system and data were operated andanalyzed using targetlynx software (waters version
4.1). The instrumental parameters were optimized during the direct infusion of the compounds with 50 %solvent (0.1 % acetic acid in water/acetonitrile at 1:1[v/v]) at a flow rate of a 5 μL/min. The [M+H] + ionsof the compounds were identified by LC-MS, with theMS1 operated in the full scanning mode in the rangeof 50 - 800 m/z. A product ion spectrum was obtainedfor each compound (Table 1). Multiple reaction monitoring (MRM) was used for the quantitative analysis.
Calibration curves - Stock solutions of indoleamine,
catecholamine and intact amino acid were used to prepare the working standards of each compound (concentrations of 1 - 5,000 μ mol/L) by serial dilution in
4:6 acetonitrile: water with 0.1 % formic acid; isotopesof the internal standards GABA-d 6 , DP-d 4 and 5-HTd 4 were prepared at a concentration of 25.0 μ mol/L.
The calibration curves of amino acids were plottedusing the absolute calibration curve method, and theconcentration ratios of the analytes to the internal
standards (GABA/GABA-d4, DA, L-DOPA, EP and
NE/DA-d 4, 5-HT, 5-HTP and HIAA/5-HT-d 4 ) as the xaxes and the peak area ratios of the analytes to theinternal standards as the y axes. The calibration curves
for each compound showed excellent linearity over theconcentration range used (R > 0.995), and the detection limits of these were shown in Table 2.
Overall method recovery - Recovery tests were performed to search for the recovery rates and assess theaccuracy of the method. Several concentration mixtures of the standards and internal GABA-d 6, DA-d 4
and 5-HT-d 4 standards were added to 100 μ mol/L.
The samples were measured and evaluated for the recovery rate and relative standard deviation (RSD %)using LC-MS/MS. The accuracy and precision of theentire analytical procedure were evaluated by spiking
the serum samples (n=3 - 5) with 100 μ mol/L. The

levels of free amino acids and neurotransmitters weresubtracted from the spiked levels of the analysis in themale rat serum. Accurate data with 70 - 120 % recovery and RSD under 25 % and limits of detection(LOD; signal / noise [S/N] > 3) were obtained, respectively, as previously indicated by the FDA as a guideline (17).
Identification of free amino acids and neurotransmitters - The HPLC system was a UFLC Nexera(Shimadzu, JPN) instrument, consisting of a vacuum
degasser, autosampler, binary pump and column oven.
The mass analyzer was a micrOTOF - QII time offlight mass spectrometer (Bruker Daltonics, Bremen,
Germany), equipped with an orthogonal electro - spray(ESI) source and a 6 - port diverter valve. The instrument was operated in the positive ion mode, using a
range of 50 - 500 m/z. The capillary voltage of the ion
source was set to 4,500 V; then, the nebulizer gas flow
was 1.6 bar and the dry gas flow was 8 L/min. The dry
temperature was set to 180 °C. Instrument calibration
was performed prior to each sequence using 10 m
mol/L sodium formate/2-propanol (1:1, v/v). The postrun internal mass scale calibration for the individual
samples was performed using data acquired during acalibration injection at the beginning of the run via a 6- port diverter valve equipped with a 20 μL loop. The
calibrant was also injected at the end of each run toverify the calibration stability. The instrument calibration and post-run internal mass scale calibrations were
performed using sodium formate ions Na (NaCOOH)
1-7 , ranging from 90.976645 to 498.901189 m/z, withan accuracy of 5 ppm. The data processing was performed using Data Analysis software (version 4.0,
Bruker Daltonics). The base mass peak (after background subtraction) was measured after the proton
subtraction in the compounds (10 ppm tolerance). For
each retrieved chemical formula, the mass error (difference between the measured and theoretical masses)
and SigmaFitTM (a parameter, were calculated using
Bruker software, accounting for the difference between the theoretical and measured isotonic pattern;
the lower the sigma value (usually < 0.05), the betterthe matching.
Results
Certification of the analytical method developed Extraction and preparation procedures of the free amino acids and neurotransmitters are shown in Fig. 1.
These compounds were extracted with acetonitrile/water (4:6) solution containing 0.1 % formic acidas the final concentration for efficient positive ionization. Standard compounds, such as deuteriumsubstituted DA, 5-HTP and GABA, were added into
the homogenates for the internal standards. Hexane
was added into the supernatant solution to removehydrophobic contaminants, as shown in Fig. 1. Highperformance liquid chromatography profiles using an
3

“Intrada Amino Acid” column without sample modification are shown in Fig. 2. For the LC-MS chromatography monitoring at 132 m/z, Leu and Ile were separated completely within 7 min with the column (Fig. 3
A and B). Because a high Ile peak was observed fromthe chromatography monitoring of 132 → 70 m/z ion
without a Leu peak (Fig. 3 A), the amino acid could bequantitatively determined. Leu peaks with a molecularmass at 132 → 43 m/z could also be determined quantitatively (Fig. 3 B). A Leu peak with 132 → 43 m/z
and Ile peak with 132 → 70 m/z were recognized as
the first target ions respectively, as shown in Table 1.
By using the present method, 20 kinds of amino acids
and neurotransmitters were completely separated within 20 min, as shown in Fig. 2, and the levels were determined in each tissue, indicating that the metabolic
relationships between amino acids and neurotransmitters consisting of amino acids can be discussed. Identification processes of amino acids, such as Leu, contained in the muscle extract are shown in Fig. 4. Bymonitoring with 132.101905 ± 0.05 Da, a dabble
peaks extracted from the muscle was detected on the
MS chromatography profile as shown in Fig. 4 B, andit had same retention time (5.80 min) with standard
Leu, as shown in Fig. 4 A, which depicts chromatogram monitoring with the same mass. After analysis of
the components with a retention time at 5.80 min, as
shown in Fig. 4 A and B, with the LC-TOF MS, theresulting LC-TOF chromatography is shown in Fig. 4.
C and D. The theoretical profile of Leu is shown in E
panel. After comparison with the theoretical value, the“MS errors” of the standard and extracted Leu were
only 5.0 and 9.2 mDa, and sigma values of them were
0.5 and 1.0, respectively, as shown in Fig. 4. C, D andE, showing that the peak obtained in panel B was identified as Leu. Identification of neurotransmitters was
performed as shown in 5-HT. The chromatography
patterns of the standard 5-HT (Fig. 5) and extracted 5HT from the rat muscle (B) were shown using LCTOF MS monitoring with 177.102239 ± 0.05 (m/z).
The mass spectrum of the peak that had a retention
time at 10.80 min in the standard shown in A panel (C)and in the extracted peak in the B panel (D) using LCTOF MS analysis. The mass spectrum of the theoretical pattern of standard 5-HT is also shown (E). A small
d-value (sigma value) was observed at 0.00188, andthe mass error was only 3.2 mDa in the D panel, which
was identified as 5-HT. Other neurotransmitters were
all identified with the SigmaFit analysis as in the figure. The recovery and LOD with a RSD are shown in
Table 2. Accurate data with 78.3 - 112.1 % recovery,

RSD with less than 5 % and LOD as previously indicated by FDA as a guideline (FDA 2001) were obtained as shown in Table 2. These data indicate that thepresent assay method is available for measuring all
free amino acids and neurotransmitters contained inorgans.
Concentration of amino acids and neurotransmitters in rat organs - Free amino acids and neurotransmitters contained in the various rat organs were assayed by the method developed in the present study,
and the content levels are shown in Table 3 and 4. The
levels of the free amino acids in the pituitary glands
were especially higher than those in the other organs(Table 4). The Gln had higher level content in all organs (m mol / L order) compared with other aminoacid, even in the blood, indicating that Gln is a final
acceptor and transporter of amino residues in the degradation of amino acids. In the data on the neurotransmitters, AC was distributed in all organs, even in
the blood, but the GABA levels were higher in thebrain, and GABA was not observed in the blood. 5-HT
was only detected in the cerebrum of the brain tissues(Table 4). And high levels of EP, NE, DA and L-DOPA
were observed in the adrenal glands (Table 3). The
BCAA content levels in the gastrocnemius muscle(GM) and extensor digitorum longus muscle (EDL)
were lower than those of the soleus muscle (SM) as
shown in Table 5. Furthermore, the BCAA content in
the GM was drastically lower than that in the EDL.
Leu, which plays important roles in muscle regeneration, mostly had a higher level in the SM.
Discussion
Progression of the method development in thisstudy - The information on the real state of metabolism was obtained by determining the free amino acids
contents. Amino acid analysis has presented an analytical challenge in terms of the sample preparation, separation, and detection. Among the separation methods,
liquid chromatography has prevailed in the amino acidanalysis field with either pre-column or post-column
labeling techniques to improve either the separation ofamino acids or selectivity and sensitivity of the analysis (13). More simplicity and ease were necessary for
automatic analysis. We developed a simultaneous determination method of free amino acids without any
derivative or labeling reactions in the rat various organs. We developed an accurate determination methodwith 70 - 120 % recovery, RSD under 5 %, the lowest
LOD, and signal/noise (S/N)>3 (Table 2). These data
were within the limits indicated by the FDA guidelines(17). A recent method was reported to have higher
4

sensitivity and selectivity when combined with postcolumn labeling techniques for amino acid analysis;
however, the chromatography process of that method
takes 200 minutes (13). The procedure developed inthis study included the preparation of samples for 40
min, a complete separation of 20 amino acids and neurotransmitters with the “Intrada Amino Acid” column
and identification by LC-TOF MS analysis within 20
min, as shown in Fig. 2. Our direct method developedin this study does not require consideration of the reagent stability, reproducibility or compatibility, which
are important risk factors in amino acid labeling techniques. The “Intrada Amino Acid” column could separate isoforms, Leu and Ile, within 7 min, as shown in
Fig. 2. Most current assay methods require the derivatization or labeling of amino acids for qualitative andquantitative analysis. For simple and fast performance,
several procedures without any derivatization or labeling of amino acids were developed (18, 19); however,these methods could not separate all of the amino acids in a chromatography performance with LOD levels.
We developed a method in this study that can simultaneously analyze all 20 amino acids by using a column“ Intrada Amino Acid” with higher sensitivities (nmol/L levels LOD). The concentration of neurotransmitters is known to fluctuate along with the current neuro-functional viability. The content levels ineach tissue provide substantial information about theirstate of health and disease. In the present study, thelevels of neurotransmitters, such as GABA, 5-HT, DA
and AC in the various organs, could also be separated
and determined on the chromatography and detectedwith the MS analysis. This method completes the simultaneous assay within 80 min and has the lowest LOD
values compared with other methods (20, 21). The
monitoring of neurotransmitters, such as AC, MLT,
GABA, 5-HT, EP, NE, DA and their metabolites, isessential to elucidating the neuronal functions andpathological states. Analysis of the neurotransmitterslevels could aid in the disease diagnosis, prognosis andmonitoring of treatments. Several HPLC or LC-MS
methods have been reported to determine the levels of
various neurotransmitters (21, 22); however, few studies have reported the simultaneous determination of allof these neurotransmitters and their metabolites in thetissues. Recently, several studies on simultaneous determination methods using LC-MS analysis were reported to have lower variability, good recovery andaccuracy (14-16). The LOD of these methods was in
the ng/mL level, corresponding to the μ mol/L levelsof neurotransmitters. In this study, we developed a

new method for measuring neurotransmitters and their
metabolites in rat tissues with a similar LOD, accuracy
and recovery, which could simultaneously be evaluated with amino acids analysis using a single column.
Local levels of amino acids and neurotransmittersin rat organs - Higher levels of Gln , Glu and Ala
were observed in the skeletal muscle compared withother amino acid, indicating that these amino acids
were tissue specific acceptors of amino residues fromthe deamination of other amino acids (Table 3). The
free amino acid levels in the skeletal muscle werelower than in other tissues except for these three amino acids (Gln, Glu and Ala), indicating an extensive
scale of amino acid metabolism in the organ (Table 3).
Two types of the neurotransmitter localization wereobserved, as shown in Table 3 and 4. GABA and AC
were detected in all tissues, and other neurotransmitters were specifically located in each tissue. The 5-HT
was only detected in the cerebrum of the brain tissues,reflecting the localization of the 5-HT neuron mainlyin the cerebrum (Table 4). There were high levels of
EP and its precursors, NE, DA and L-DOPA, in theadrenal gland (Table 3), which was certainly from thepresence of the synthetic epinephrine system in theglands. The reason for the higher level of GABA in the
cerebellum was clearly because of the high number of
GABA neurons in that region. The higher level of 5HT in the skeletal muscles coincided with the localization of the 5-HT2A receptor in the tissue, where 5-HT
might act as an intracellular signal (23), and facilitatemuscle spasms after spinal cord injury (24).
BCAA in the muscle types - The levels of BCAA in
the GM and EDL were lower than in the SM (Table 5).
The levels are inversely correlated with the content
rates of fast muscle (Type II), which accounts for approximately 60 - 100 % of the GM, 96 % of the EDL
and 2.7 % of the SM (25). This may be reflected by
the properties of fast muscle, which has a higher turnover rate and frequently incorporates BCAA into themuscle proteins as a result of the higher glucocorticoid
receptor (GR) expression (26). It is interesting, though
the cause is unknown, that the BCAA content in the
GM was drastically lower than that in the EDL, suggesting that BCAA has important roles in protein metabolism or muscle regeneration. A higher level of Leu
was found in the SM, which consists of slow typemuscle (Table 5). High Leu levels can inhibit protein
degradation in skeletal muscles and induce muscle
protein synthesis by enhancing its sensitivity to insulin(27). The degradation rate of muscle proteins is slow
due to the lower expression of the GR in the SM (26).
5A higher level of Leu might be involved in the slow
turnover of the muscle protein in the SM.
Conclusion - Evaluation of the tissue-specific localization and concentration of amino acids and neurotransmitters was performed using the method developed in this study. Several new observations on themetabolisms of amino acids, such as BCAA, and neurotransmitters in the skeletal muscle were made.

Acknowledgements
The authors are grateful to Mr.Itaru Yazawa of Imtakt
Corporation, Kyoto JPN who provided helpful comments and suggestions for “Intrada Amino Acid column”. And the authors are thankful to Mr.Michio Sasaki of Japan Meat Science and Technology Institutefor his helpful.

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Table 1. MS/MS conditions in SRM transitions employed for the determination of amino acids
and neurotransmitters.
CompoundQuantitative ion
Product ion
Collision energy(m/z )(CE)First target ion
Product ion
Collision(m/z )
energy (CE)

Precursor ion(m/z )

Cone voltage(CV)

Alanine (Ala)

90

20

44

10

-

-

Arginine (Arg)

175

30

70

20

116

10

Asparagine (Asn)

133

20

74

10

46

20

Aspartic acid (Asp)

134

30

88

10

74

10

Cystine (Cys)2

241

20

120

20

152

10

Glutamine (Gln)

147

20

84

15

130

10

Glutamic acid (Glu)

148

20

85

10

131

10

Glycine (Gly)

76

30

30

10

-

-

Histidine (His)

156

30

110

10

83

20

Isoleucine (Ile)

132

20

70

20

86

10

Leucine (Leu)

132

20

43

20

30

10

Lysine (Lys)

147

20

84

20

130

10

Methionine (Met)

150

20

56

20

61

20

Phenylalanine (Phe)

166

20

103

30

120

10

Proline (Pro)

116

30

70

10

43

20

Serine (Ser)

106

20

60

10

42

20

Threonine (Thr)

120

30

74

10

56

10

Tryptophan (Trp)

205

20

118

30

91

40

Tyrosine (Tyr)

182

20

136

10

91

30

Valine (Val)

118

20

72

10

55

20

4-Aminobutanoic acid (GABA)

104

20

87

10

69

15

Acetylcholine (AC)

146

30

87

14

43

24

Dopamin (DA)

154

20

137

10

91

20

Serotonin (5-HT)

177

20

160

10

132

30

Epinephrine (EP)

184

20

166

10

107

20

Norepinephrine (NE)

170

20

152

10

107

20

5-Hydroxytryptphan (5-HTP)

221

30

204

10

162

20

5-Hydroxyindoleacetic Acid (5-HIAA)

191

30

146

10

91

30

L-3,4-dihydroxyphenylalanine (L-DOPA)

198

20

152

10

107

30

Melatonin (MLT)

233

30

174

10

159

30

Isotope
GABA d6

110

20

93

10

49

20

DA d4

158

10

95

20

141

10

5-HT d4

181

20

136

20

118

30

Table 2. Recovery tests of additive indoleamine, catecholamine and intact amino acidinto the serum.

Compound
Ala
Arg
Asn
Asp
(Cys)2
Gln
Glu
Gly
His
Ile
Leu
Lys
Met
Phe
Pro
Ser
Thr
Trp
Tyr
Val
GABA
AC
DA
5-HT
EP
NE
5-HTP
5-HIAA
L-DOPA
MLT

Serum (1.0 ml)

LOD (nmol/L)

Recovery(%)

RSD(%)

84.5

1.0

0.21

96.3
88.8
101.5
95.9
86.0
92.0
87.2
78.3
96.8
87.6
83.2
83.6
95.1
91.2
84.5
90.5
84.7
78.5
100.1
100.4
93.0
96.3
100.1
110.3
91.7
112.1
90.0
101.7
98.9

4.7
3.1
3.9
1.5
4.0
3.2
2.5
0.5
0.7
1.7
4.1
1.8
2.7
1.5
2.7
4.9
4.5
0.5
0.4
4.1
3.4
3.5
0.8
2.8
3.5
2.0
4.2
3.0
4.6

0.18
2.06
2.26
2.37
0.11
0.99
3.62
0.34
1.17
0.51
0.07
0.13
0.48
0.23
0.46
0.26
0.21
0.35
0.28
2.00
9.20
61.6
6.51
8.78
41.4
17.0
0.27
61.2
0.11

LOD: limits of detection
RSD: Relative Standard Deviation
Data presented mean values of n=3 - 5
Recovery data obtained were shown with relative standard deviation (RSD).
The accurate data having 70 - 120 % recovery and RSD under 25 % and limits ofdetection (LOD; signal / noise [S/N]>3) were obtained respectively as previously
indicated by FDA as a guideline (FDA 2001). The accuracy and precision of theentire analytical procedure were evaluated by spiking the serum samples (n=3 5) with 100 μ mol / L.

3.2
193
.

10.5

25
10
1.7
1.3
0.82
1.40E+03
13
9
2.4
8
10
22
4.0
7.1
18
5
13
7
7
6

Serum

612 ±
232 ±
67.5 ±
30.1 ±
3.10 ±
5.20E+04 ±
140 ±
225 ±
46.3 ±
165 ±
153 ±
417 ±
61.8 ±
89.4 ±
485 ±
145 ±
260 ±
150 ±
127 ±
207 ±
–
63.7 ±––
56.0 ±
636 ±
–
–
–
–

978
404
72.8
440
8.00
2.60E+04
1.00E+03
1.00E+03
141
154
366
490
183
299
765
484
390
76.7
241
142
53.5
578

±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
–
–
–
–
–
–
–
–

Testis
95
47
19.8
57
4.02
1.01E+03
1.02E+02
1.11E+02
16
30
55
95
28
37
97
72
74
12.1
37
12
15.4
50

55.6

47.8

444
376
367
251
6.20
1.46E+04
299
980
249
328
584
587
290
452
272
545
382
95.0
328
78.3
15.4
1.14E+04

±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
–
±
–
±
–
–
–
–

Liver

10.8

13.7

263
121
89
40
1.20
3.80E+03
63
169
69
50
104
130
56
94
82
104
85
23.3
66
19.6
9.9
4.18E+02

87.6

71.7

±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
–
±
–
±
–
–
–
–
17.3

18.3

36
28
2.25E+02
1.32E+0273
1.60E+02
1.05E+02
2.32E+02
1859
1.21E+02
29
38
11
34
1.38E+02
36
20
13
3.2
17
3.09E+02

Kidney521
860
1.27E+03
1.14E+03
297
1.62E+04
1.32E+03
3.11E+03245
856
1.15E+03
551
479
579
556
1.10E+03
448
241
273
78.1
166
2.12E+03

255

±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
–
–
–
±
–
55

9.03E+02
2.04E+02
165
49
2.6
5.07E+03
3.00E+02
7.08E+0230
112
164
1.45E+02
95
144
3.12E+02
94
163
44
176
192
5.0
22.4
6.31E+03
1.36E+04
5.02E+03

Adrenal glands
4.34E+03
1.19E+03
394
776
12.1
2.11E+05
1.89E+03
2.13E+03
123
444
926
2.23E+03
184
763
3.05E+03
592
887
220
570
858
60.0
48.2
6.27E+04
5.52E+04
3.24E+04

Concentration of amino acids and neurotransmitters in rat organs were assayed by the method developed in this study as described in "materials and methods".
Data were shown as the means ± S.D. for 5 animals (μ mol / L or g of tissue weight). “ – ” mean as “LOD”.

Ala
Arg
Asn
Asp
(Cys)2
Gln
Glu
Gly
His
Ile
Leu
Lys
Met
Phe
Pro
Ser
Thr
Trp
Tyr
Val
GABA
AC
EP
NE
DA
5-HT
5-HTP
5-HIAA
L-DOPA
MLT

CompoundTable 3. Amino acids and neurotransmitters in various tissues of adult male rats.

0.16

444

30.5

±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
–
±
–
±
–
–
–
±

0.08

156

6.6

3.10E+02
21
1.3
2.3
12.2
8.03E+02
4.20E+02
52
6.2
9.0
10.0
5.9
2.4
11.5
11
26
23
3.2
8.4
6.0
0.54
1.02E+02

Skeletal muscle
1.80E+03
112
24.1
34.9
51.1
9.70E+03
2.80E+03
783
95.0
41.7
56.6
36.8
24.3
66.7
303
186
209
14.2
59.9
70.9
4.40
1.01E+03

Table 4. Amino acids and neurotransmitters in brain tissues of adult male rats.
Compound
Ala
Arg
Asn
Asp
(Cys)2
Gln
Glu
Gly
His
Ile
Leu
Lys
Met
Phe
Pro
Ser
Thr
Trp
Tyr
Val
GABA
AC
EP
NE
DA
5-HT
5-HTP
5-HIAA
L-DOPA
MLT

Brain
Cerebrum
935
420
83.2
2.11E+03
16.7
2.74E+04
2.67E+03
676
165
168
267
299
137
266
742
541
557
64.2
183
182
929
3.59E+03
104
66.4

0.09

±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
–
±
–
±
–
–
–
±

143
76
19.9
4.02E+02
4.4
3.60E+02
4.20E+02
77
37
40
54
43
44
43
157
101
96
15.6
58
34
181
2.01E+02
14

Cerebellum
2.30E+03793
124
2.30E+03
44.8
1.33E+05
5.10E+03
1.02E+03
107
264
434
571
176
521
1.89E+03
651
940
142
344
444
2.97E+03
2.46E+03
90.0

18.9

0.03

0.20

±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
–
±
–
–
–
–
–
±

3.36E+02
109
28
2.08E+02
12.2
9.40E+02
7.37E+02
2.08E+02
30
74
123
96
69
75
4.20E+02
99
158
22
30
78
3.09E+02
2.45E+02
51.7

0.02

Hipocampus
2.52E+03
618
180
2.97E+03
27.2
2.09E+05
6.03E+03
977
73.7
287
343
433
102
444
2.03E+03
869
984
120
362
520
3.48E+03
427

±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
–
–
–
–
–
–
–
–

5.07E+02
87
27
1.40E+02
4.2
3.87E+02
9.74E+02
205
20.1
27
24
62
16
57
2.04E+02
60
206
24
72
53
2.78E+02
35

Pituitary glands
2.12E+03
1.64E+03
2.01E+03
4.62E+03
190
1.90E+05
1.78E+03
1.21E+03274
2.22E+03
3.43E+03
3.84E+03888
5.89E+03
3.47E+03
3.96E+03
4.16E+03
1.21E+03
3.13E+03
5.69E+03
1.17E+03
203

1.82E+03

±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
–
–
±
–
–
–
–
–

Concentration of amino acids and neurotransmitters in rat brain were assayed by the method developed in this study as described in "materials andmethods". Data were shown as the means ± S.D. for 5 animals (μ mol / L or g of tissue weight). “ – ” mean as “LOD”.

1.20E+03
6.21E+02
7.01E+02
9.05E+02
52
5.02E+03
6.60E+03
5.70E+03
47
1.51E+03
1.31E+03
1.15E+03264
1.87E+03
1.08E+03
2.42E+03
2.27E+03
5.07E+02
1.50E+03
2.20E+03
8.07E+02
93

4.04E+02

Table 5. Contents of BCAA in rat muscle type and Tukey-Kramer Multiple Comparisons
BCAA Contents (n mol / g of Tissue)
Total
Muscle type

Leu

Ile

Val

Soleus muscle (SM)410.8 ± 86.6

261.8 ± 63.1

266.3 ± 33.7

938.8 ± 182.1

Extensor digitorum longus muscle (EDL)

246.2 ± 21.2

155.7 ± 12.1

214.3 ± 20.7

616.2 ± 53.6

Gastrocnemius muscle (GM)

24.0 ± 0.9

17.1 ± 0.5

14.8 ± 0.5

55.3 ± 2.7

Leu

Ile

Val

Tukey-Kramer multiple comparisonsMuscle type
GM：SM
GM：EDL
SM：EDL

Absolute Difference
15.03**
8.64**
6.4*

13.20**
7.47*
5.72*

22.04**
17.49**
4.55*: p < 0.05**: p < 0.01

Extraction procedure
Weighing of tissue
added per gram of tissue by adding 4.0 mL solution(acetonitrile : water with 0.1% formin acid ; 4:6, v/v)
added 1.0 mL of 100 μ mol / L DA-d4, 5-HT-d4 and GABA-d6
homogenized with biotechnology mixer
centrifuged at 8,000 rpm (10,160 ×g) for 20 min
Supernatant
1.0 mL of supernatant
added 1 mL of hexane (saturated with acetonitrile)
mixed by vortex for 10 sec and centrifuged at 3,000 rpm (700 ×g) for 5 min
filtrated by centrifugation at 12,000 rpm (11,400 ×g) for 5 min(Ultra free-MC 0.22 μm pore size, Millipore)Sample for LC-MS/MS and LC-TOF MS analyses

Fig. 1
Preparation procedure for the MS analyses from rat organs
Amino acids and catecholamine were extracted from rat organs with acetonitrile solution, andthe internal standards (DA, 5-HT and GABA transferred 4 molecules of hydrogen to 4 ofdeuterium (DA-d4), 4 of hydrogen (5-HT-d4) and 6 of hydrogen (GABA-d6), respectively)
were added into the preparations.

Fig. 2
Separation of amino acids and neurotransmitters by LC-MS using an “Intrada Amino Acid” col-umn
The quantitative ions of each compound determined in Table 1 were monitored with positive ion modescanning of a gradient mobile phase, as described in the Materials and Methods. All compounds were analyzed within 20 min by the analytical method.

13

Ile

A)

Intensity

132>70B)

Leu

132>43Fig. 3
Separation of Leu and Ile by LC-MS using “Intrada Amino Acid” column
Standard Leu and Ile were separated completely within 7 min. The positive ions having 70 m/z
derived from precursor Ile and 43 m/z peak derived from Leu showed a single maximum peak,
respectively (A,B). The quantitative and first target ions of the all compounds tested determined as
showing in the figure and listed in Table 1.

A

B

Intens.
x105
2.0

Amino 100nmol ml_1-1_01_2924.d: +MS, 6.0min #214

C

1.5
1.0

Meas. m/z
132.102569

Ion FormulaC6H14NO2

m/z
132.101905

err [ppm]-5.0

Meas. m/z
132.103118

Ion FormulaC6H14NO2

m/z
132.101905

err [ppm]-9.2

mSigma
0.5

0.5
0.0
x104

6

blood 1_1-4_01_2929.d: +MS, 5.9min #213

D

4

mSigma
1.1

2

0
x104
6

blood₁₁-4₀1₂929.d:C₆H₁₄NO₂, 132.1019

E

4

2

0
132.00132.25132.50132.75133.00133.25133.50133.75134.00134.25 m/z

Fig. 4
Identification of Leu by LC-TOF MS analysis
The chromatography patterns of the standard Leu (A) and extracted Leu from the rat
muscle (B) were shown using LC-TOF MS monitoring with 132.101905 ± 0.05 (m/z). The
mass spectrum of the peak that had a retention time at 5.80 min in the standard shown in A
panel (C) and in the extracted peak in the B panel (D) using LC-TOF MS analysis. The
mass spectrum of the theoretical pattern of standard Leu is also shown (E). A small d-value(sigma value) was observed at 0.00011, and the mass error was only 9.2 mDa in the Dpanel, which was identified as Leu. Other compounds were all identified with the SigmaFit
analysis as in the figure.

A

B

C

D

Meas. m/z
177.104485

Ion Formula
C10H13N2O

m/z
177.102239

err [ppm]-12.7

mSigma15.5

Meas. m/z
177.096572

Ion Formula
C10H13N2O

m/z
177.102239

err [ppm]
3.2

mSigma18.8

EFig. 5
Identification of 5-HT by LC-TOF MS analysis
The chromatography patterns of the standard 5-HT (A) and extracted 5-HT from the rat
muscle (B) were shown using LC-TOF MS monitoring with 177.102239 ± 0.05 (m/z). The
mass spectrum of the peak that had a retention time at 10.8 min in the standard shown in A
panel (C) and in the extracted peak in the B (D) panel using LC-TOF MS analysis. The
mass spectrum of the theoretical pattern of standard 5-HT is also shown (E). A small dvalue (sigma value) was observed at 0.00188, and the mass error was only 3.2 mDa in the
D panel, which was identified as 5-HT. Other compounds were all identified with the
SigmaFit analysis as in the figure.