detection of 28 neurotransmitters and related compounds in biological fluids by liquid...

RAPID COMMUNICATIONS IN MASS SPECTROMETRY

Rapid Commun. Mass Spectrom. 2006; 20: 1405–1421

) DOI: 10.1002/rcm.2459
Published online in Wiley InterScience (www.interscience.wiley.com
Detection of 28 neurotransmitters and related compounds

in biological fluids by liquid chromatography/tandem

mass spectrometry

Sophie Bourcier1*, Jean-Francois Benoist2, Frederic Clerc2, Odile Rigal2,

Meryam Taghi1 and Yannik Hoppilliard1

1Laboratoire des Mecanismes Reactionnels, Unite Mixte de recherche CNRS 7651, Ecole Polytechnique, 91128 Palaiseau Cedex, France2Service de Biochimie-Hormonologie, Hopital Robert Debre APHP, 75019 Paris, France

Received 12 December 2005; Revised 25 February 2006; Accepted 27 February 2006

*CorrespoPolytechnE-mail: so

This work presents two liquid chromatography/tandem mass spectrometry (LC/MS/MS) acquisition

modes: multiple reaction monitoring (MRM) and neutral loss scan (NL), for the analysis of 28

compounds in a mixture. This mixture includes 21 compounds related to the metabolism of three

amino acids: tyrosine, tryptophan and glutamic acid, two pterins and five deuterated compounds

used as internal standards. The identification of compounds is achieved using the retention times

(RT) and the characteristic fragmentations of ionized compounds. The acquisitionmodes used for the

detection of characteristic ions turned out to be complementary: the identification of expected

compounds only is feasible by MRM while expected and unexpected compounds are detected by

NL. In the first part of this work, the fragmentations characterizing each molecule of interest are

described. These fragmentations are used in the second part for the detection by MRM and NL of

selected compounds in mixture with and without biological fluids. Any preliminary extraction

precedes the analysis of compounds in biological fluids. Copyright # 2006 John Wiley & Sons, Ltd.

We present liquid chromatography/tandem mass spectrom-

etry (LC/MS/MS)methods for the analysis of 28 compounds

in a mixture. This mixture includes 26 compounds (five are

deuterated compounds used as internal standards) belong-

ing to three of the most important families of neurotrans-

mitters. These compounds are the result of the normal

metabolism of three amino acids: tyrosine, tryptophan and

glutamic acid, precursors of catecholamines, serotonine and

g-aminobutyric acid, respectively.1 Two pterins, biopterin

and neopterin, precursors of tetrahydrobiopterin (BH4), are

also studied. BH4 is the essential cofactor of tyrosine and

tryptophan hydroxylases, two rate-limiting enzymes for

catecholamine and serotonine biosynthesis.1

The neurotransmitters are involved in a variety of

regulating systems such as stress and learning, and also in

the control of many processes of metabolism and in the

immune system.2 Until now, analysis of these compounds

has been routinely carried out using high-performance liquid

chromatography (HPLC) coupled with electrochemical3–6

and fluorescence7–10 detection.

However, these methods are time-demanding and often

require derivatization of analytes and/or large quantities of

sample. Analyses using capillary electrophoresis (CE) with

UV or mass spectrometry (MS) detection have also been

reported.11–15 LC and CE coupled with tandem mass

spectrometry (MS/MS)16–24 have been used to increase the

ndence to: S. Bourcier, DCMR, UMR CNRS 7651, Ecoleique, 91128 Palaiseau Cedex, [email protected]

specificity of analyses. Recently, mixtures of few catechol-

amines in brain tissue17,18 and in urine19–21 were analyzed by

LC/MS/MS with good sensitivity. Quantitation of indola-

mines in the rat brain by LC/MS/MS22 allows the evaluation

of in vivo inhibition of rat brain monoamine oxidases.

Identification of serotonin and its precursors in human

plasma was carried out by capillary zone electrophoresis

time-of-flight mass spectrometry (CZE-TOFMS).14 In the

family of glutamic acids, homocarnosine,23 GHB16 and

GABA24 were separately studied by electrospray ionization

(ESI)-MS/MS. Finally, a few results have been published

concerning the study of pterins in biological fluids by MS.

Pterin was identified in human urine by reversed-phase ion-

pairing chromatography coupled with ESI-MS.25 Thus, as far

as we know, LC/MS/MS techniques have been used for the

simultaneous analysis of only a limited number of com-

pounds in the serotonine and catecholamines families.

In many cases, clinical signs of metabolism disorders

cannot be related to a deficiency or a surfeit of compounds

known to be involved in the metabolism. The goal of this

work was not the quantitation of compounds, but the

identification of compounds associated with these clinical

signs. These compounds could be quantified, later, if

necessary.

At the beginning of this work, the conditions allowing the

simultaneous identification of compounds of interest present

Copyright # 2006 John Wiley & Sons, Ltd.

1406 S. Bourcier et al.

in a reference mixture were determined. Each compound

was analyzed alone in acidified water and characterized

by diagnostic MS/MS transitions unambiguously allowing

its identification, in a mixture. These diagnostic tra-

nsitions are used to record reference profiles related to

known mixtures of neurotransmitters, in acidified water.

Fragmentation processes of some neurotransmitters were

extensively26–28 studied earlier, others are proposed here.

The prominent fragmentations are retained for the

unambiguous identification of compounds by multiple

reaction monitoring (MRM) detection. Only few neutral

losses (NH3; H2O; H2OþCO. . .) are associated with

the characteristic transitions. Such a neutral loss (NL) could

be used for the identification of new metabolites (NL

detection).

The matrix effect is then evaluated, by loading biological

fluids (urine, cerebrospinal fluid (CSF) and amniotic fluid

(AF)) with a mixture of deuterated neurotransmitters, using

the MRM detection mode. We demonstrate that we can

detect neurotransmitters in crude biological fluids. A

comparison between normal and pathological profiles

illustrates the interest of this work.

EXPERIMENTAL

ChemicalsDL-Tryptophan (Trp), 5-hydroxy-DL-tryptophan (5-HTP),

5-hydroxytryptamine hydrochloride (serotonin, 5-HT),

5-hydroxyindole-3-acetic acid (HIAA), g-aminobutyric acid

(GABA), DL-tyrosine (Tyr), DL-3,4-dihydroxyphenylala-

nine (DL-DOPA), dopamine hydrochloride (DA), DL-

norepinephrine hydrochloride (NE), 3,4-dihydroxy-

a-(methylaminomethyl)benzyl alcohol (epinephrine, E), 3-

methoxytyramine hydrochloride (3-MT), 3,4-dihydroxyphe-

nylacetic acid (DOPAC), DL-normetanephrine hydrochlo-

ride (NMN), DL-metanephrine hydrochloride (MN),

homovanillic acid, (HVA), vanillomandelic acid (VMA),

4-hydroxy-3-methoxyphenylglycol (MHPG) sulfate potass-

ium salt, g-hydroxybutyric acid sodium salt (GHB), g-

aminobutyryl histidine (homocarnosine, HC), glutamic acid

(Glu), succinic acid (SA), 2-amino-4-hydroxy-6-(1,2-dihy-

droxypropyl)pteridin (biopterin, Bio), D-erythro-neopterin

(neopterin, Neo), formic acid, and HPLC-grade solvents

were purchased from Aldrich Chemical Co. (Saint Quentin

Fallavier, France). L-Tryptophan 20,40,50,60,70-d5 (Trp-d5),

4-hydroxyphenylacetic 2,2-d2 acid (HVA-d2), GABA

2,2,3,3,4,4-d6 (GABA-d6), DL-DOPA-2,5,6-d3 (DL-DOPA-

d3) and tyrosine-2,3,5,6-d4 (Tyr-d4) were purchased from

Cluzeau (Puteaux, La Defense, France).

Sample preparationSolutions of each compound were prepared in water at

10�3M and stored at �808C. For the identification of

diagnostic transitions, these solutions were diluted at

10�5M in a mixture of H2O/CH3CN (50:50) acidified with

formic acid (0.1%) and infused into the ionization source

with a syringe pump at 10mL min�1.

An equimolar mixture of all compounds, diluted at 10�6M

with acidic water (formic acid 0.1%) (reference solution), was

used for the registration of reference profiles.


The matrix effect was studied by using three biological

fluids without any preliminary extraction of compounds:

400mL of each matrix were spiked with 100mL of reference

solution. These solutions of spiked biological fluids are

identified as: CSFS, UrineS or AFS. They were centrifuged at

3500 g for 15min at 48C. Supernatants were isolated and

evaporated to dryness under nitrogen. The dry extracts were

diluted with 200mL of acidic water. The salt effect was

evaluated using 400mL of physiological serum in place of

400mL of biological fluids. The sodium concentration in

physiological serum and in CSF is approximately the same

(150mM).

Chromatographic and massspectrometric conditionsAll samples were analyzed using a model 1100 liquid

chromatograph from Agilent Technologies (Massy, France)

coupled with an API 3000 triple quadrupole mass spec-

trometer (Applied Biosystems/MDS-Sciex, Concord, ON,

Canada). The liquid chromatograhwas equippedwith an on-

line degassing system, a binary pumping system, an

autosampler and a Valco valve with two outlets. All modules

are controlled by Analyst software, version 1.4 (Applied

Biosystems/MDS-Sciex). The mass spectra were recorded

with Q1 and Q3 working at unit or low mass resolution.

A full-scan mass spectrum of DL-DOPA-d3 was also

recorded on a Quattro II mass spectrometer (Micromass,

Waters, Manchester, UK) in order to obtain a mass

resolution better than was available with the API 3000

mass spectrometer.

Several C18 analytical columns were tested. We finally

opted for an Atlantis C18 column (2.1mm� 150mm, 5mm;

Waters, Saint-Quentin-en-Yvelines, France) equipped with

the corresponding pre-column. Salts included in crude

biological fluids were sent to waste through the Valco valve

at the beginning of separation (0–1.9min). The HPLC

solvents were water with 0.1% formic acid (A) and

acetonitrile (B). The following program of linear gradient

elution was applied: 98%A for 3min and 98–80%A from 3 to

16min. Then, before the next injection, the column was

reconditioned with 98% of A for 14min. The effluent was

introduced, without splitting, at 0.2mL �min�1 into a

TurboIon spray interface. The source temperature was

5008C. Each injected volume was 10mL of solution.

Nitrogen was used as curtain gas (CUR), nebulizer gas

(NEB), turbo gas and collision gas (CAD). Experimental

parameters fitted for the acquisition of data in bothMRMand

NL modes are summarized in Table 1.

Diagnostic transitionsThe choice of diagnostic transitions, allowing unambiguous

identification of compounds in a mixture, depends on the

energetic conditions of a fragmentation. The energetic

conditions required for an optimal detection of ions is

obtained from a breakdown graphs. For example, Fig. 1

shows the breakdown graph of protonated dopamine

(DAHþ). At low collision energy (12<CE< 26 eV), the most

abundant product ion is m/z 137 while, at higher collision

energy (CE> 35 eV), m/z 91 is prominent.


DOI: 10.1002/rcm

0

20

40

60

80

100

120

50454035302520151050(eV)Collision energy

Abu

ndan

ce%

m/z 91m/z 119m/z 137m/z 154

Figure 1. Breakdown graph of DAHþ (m/z 154) (DP¼ 14 V, CE¼ 0–46 eV).

Table 1. Experimental parameters used with MRM and NL acquisition modes

Modes IS (V) NEB (psi) CUR (psi) CAD(psi) DT(ms) DP(V) CE(eV)

Mode (þ)MRM 3500 10 12 4 100 10 to 60 10 to 40Neutral loss 3500 10 12 4 � 20 to 40 20

Mode (�)MRM �4500 12 10 4 150 �20 to �64 �10 to �35Neutral loss �4500 12 10 4 � �15 to �30 �10 to �30

IS: Ion spray, NEB: nebulizer gas, CUR: Curtain gas, CAD: Collision gas, DP Declustering potential, DT: Dwell time and CE: Collision energy(laboratory frame).�Neutral loss scans were obtained by scanning from 100 to 300 m/z in 800ms.

Detection of neurotransmitters by ESI-MS/MS 1407

The detection will be optimal using the transitions m/z

154! 137 around CE¼ 15 eV and m/z 154! 91 around

CE¼ 35 eV.

RESULTS AND DISCUSSION

This work is divided into five parts. The fragmentations of

each compound are described in the first part and the main

transitions are selected for the identification by MRM of each

molecule included in the mixture. Neutral losses characteriz-

ing families of compounds are selected in the second part. In

the third part, we present reference profiles of known

mixtures of neurotransmitters in acidic water, recorded using

both MRM and NL acquisition modes. The matrix effect is

evaluated in the fourth part, by loading biological fluids with

deuterated compounds. In the final part, the compounds of

interest are detected in crude matrices. The focus of this

qualitative study is illustrated by a comparison between

neurotransmitter profiles of normal and pathological urines.

Main fragmentations of neurotransmittersAll of the compounds with an amine function are studied in

the positive ion mode. The others are analyzed in the

negative ion mode.

The schemes presented in this part summarize the

decomposition processes of compounds of interest. They

allow us to rapidly visualize the diagnostics transitions

retained for the identification of these compounds either by

MRM or by NL. In these schemes, plain arrows and m/z

values in bold indicate diagnostic transitions for MRM


detection while boxed small molecules correspond to neutral

losses retained for the identification of families of com-

pounds.

Tyrosine and catecholaminesThe decomposition processes of protonated tyrosine, pre-

cursor of catecholamines, have been studied previously.29,30

TyrHþ, as with other protonated aromatic amino acids,

eliminates NH3 or [H2OþCO] competitively and succes-

sively. The [MH–NH3]þ ion also loses ketene (CH2CO). One

mechanism associatedwith this fragmentationwas proposed

earlier in the case of protonated phenylalanine (PheHþ)30

(Scheme 1). No scrambling is associated with NH3 and

[H2OþCO] losses from protonated Tyr-d4.

The catecholamines analyzed in the positive ion mode

have the following structures:

Y

X

HO

R1 NHR2

R1 = OH; OCH3

X = H; OH

R2 = H; CH3

Y = H; COOH

Some catecholamines are isobaric (E and NMN; DL-DOPA

andMN). In the case of co-elution, the choice of characteristic

fragmentations requires special attention. As explained in

the Experimental section, the transitions giving the most

abundant ions are preferred. The transitions retained for the

characterization of catecholamines are summarized in

Table 2.


DOI: 10.1002/rcm

Table 2. Transitions and neutral losses characterizing protonated Tyr, Tyr-d4, catecholamines and DL-DOPA-d3. In bold,

transitions and neutral losses used for the identification of compounds in the mixture

Compounds R1 X R2 Y Transitions Neutral losses DP� (V) CE�� (eV)

Tyr 182! 165 17 (NH2R2) 16 14MHþm/z 182 H H H CO2H 165! 119 46 (H2OþCO)

165! 123 42 (CH2CO)182! 136 46 (H2OþCO) 16 27

Tyr-d4 186! 169 17 (NH2R2) 16 14MHþm/z 186 H H H CO2H 186! 140 46 (H2OþCO)

DA 154! 137 17 (NH2R2) 14 15MHþm/z 154 OH H H H 137! 119 18 (R1H)

137! 91 46 (R1HþCO)154! 91 63 (NH2R2þR1HþCO) 14 34

3-MT 168! 151 17 (NH2R2) 14 15MHþm/z 168 OCH3 H H H 151! 119 32 (R1H)

168! 91 77 (NH2R2þR1HþCO) 14 36

DL-DOPA 198! 181 17 (NH2R2)MHþm/z 198 OH H H CO2H 198! 180 18 (H2O)

181! 135 46 (H2OþCO)198! 152 46 (H2OþCO) 14 15198! 139 59 (NH2R2þCH2CO) 14 40

DL-DOPA-d3 201! 154 47 (DHOþCO) 31 21MHþm/z 201 OH H H CO2H 201! 141 60 (NH2DþCH2CO)

NE 170! 152 18 (XH) 14 10MHþm/z 170 OH OH H H 152! 135 17 (NH2R2)

152! 134 18 (R1H)152! 107 45 (NH2R2þCO) 40 25152! 106 46 (R1HþCO)

E 184! 166 18 (XH) 14 12MHþm/z 184 OH OH CH3 H 166! 148 18 (R1H)

166! 120 46 (R1HþCO)166! 135 31 (NH2R2)166! 107 59 (NH2R2þCO) 30 26

NMN 184! 166 18 (XH) 14 12MHþm/z 184 OCH3 OH H H 166! 149 17 (NH2R2) 40 22

166! 134 32 (R1H)

MN 198! 180 18 (XH) 14 14MHþm/z 198 OCH3 OH CH3 H 180! 165 15 (CH3) 57 26

180! 148 32 (R1H)180! 149 31 (NH2R2)

�DP: Declustering potential,��CE: Collision energy (laboratory frame). Optimal values for characteristic transitions.


Catecholamines with X¼H (DA, 3MT, DL-DOPA)The fragmentations of 3-MTHþ and DAHþ, studied in detail

earlier,27,28 are summarized in Scheme 2. At CE¼ 15 eV, for

3-MTHþ and DAHþ (Y¼R2––H), the loss of NH3 is

prominent. At CE¼ 35 eV, the main fragmentation corre-

sponds to the successive losses of NH3, R1H (R1H––H2O or

CH3OH) and CO from [MH]þ.

DL-DOPAHþ (Y¼COOH and R2–– H) fragments similarly

to TyrHþ (vide supra), losing NH3 and [H2OþCO] competi-

HO

NH3

COOH

[TyrH-H2O

[TyrH-NH3

TyrH+

- NH3

- H2O- COm/z 182

m/z 165

m/z 13

Scheme 1. Decompo


tively and successively; then [MH–NH3]þ loses CH2––CO

(Scheme 3). In the present case, the last fragmentation, much

more important than in the case of tyrosine, has been selected

as the diagnostic transition.

The fragmentation mechanisms of DL-DOPAHþ have

been studied by means of theoretical calculations and the

results will be published soon.

The mass spectrum of protonated DL-DOPA-d3, used for

the study of the matrix effect in biological fluid, shows a

-CO]+

]+ [TyrH-NH3-CH2=CO]+- CH2CO

6 - NH3

- H2O - CO

[TyrH-H2O-CO-NH3]+

m/z 123

m/z 119

sition of TyrHþ.


DOI: 10.1002/rcm

[MH-H2O-CO]+

[MH-NH3]+[MH-NH3-CH2=CO]+

COOH

NH3

HO

HO

DL-DOPAH+

m/z 198

m/z 181

m/z 152

m/z 139- NH3

- H2O- CO

- CH2CO

- NH3

- H2O

m/z 180 [MH-H2O-CO-NH3]+

m/z 135

- H2O- CO

Scheme 3. Decomposition of DL-DOPAHþ.

OH

NH3R1

R1=OH (DA); OCH3(3-MT)MH+

[MH - NH3]+[MH - NH3 - R1H]+

[MH - NH3 - R1H - CO]+

- R1H- NH3

- CO

DA m/z 1373-MT m/z 151

DA, 3-MT m/z 91

DAH+ m/z 154; 3-MTH+ m/z 168

DA, 3-MT m/z 119

Scheme 2. Decomposition of DAHþ and 3-MTHþ.

0

100154

153

183

155

182

201

184

200190170150 m/z

(%)

Abu

ndan

ce

NH- 3

DNH- 2

CODOH --

H- 2 COO -

-D2 COO - D- 2NH

Figure 2. Partial collision mass spectrum of [DL-DOPAH-þ


scrambling associated with the eliminations of NH3 and

[H2OþCO]. This scrambling involves six atoms including the

hydrogens of NH2 and COOH, the protonating hydrogen

and both deuterium atoms in the ortho position (Fig. 2). The

diagnostic transition corresponds to the successive losses of

DOH and CO from the protonated molecule.

Catecholamines with X¼OH (NE, E, NMN, MN)At low collision energy (CE¼ 12 eV), these protonated

compounds eliminate H2O giving a very stable carbocation

stabilized by the aromatic ring. The decomposition processes

of [MH–H2O]þ occurring at higher collision energy are

included in Scheme 4. The fragmentation mechanisms of

NEHþ and NMNHþ have been extensively described
d3] (m/z 148–210).
MH+

NH2R2

HO

OHR1

[MH-H2O-R1H]+

[MH-H2O]+

[MH-H2O-R1H-CO]+

[MH-H2O-NH2R2-CO]+

[MH-H2O-NH2R2]+

- NH2R2

- R1HY=R2=H, R1=OH (NE),OCH3(NMN)Y=H; R2=CH3, R1=OH (E); OCH3 (MN)

- NH2R2- CO

- R1H- CO

- CH3.

MN m/z 165

- H2O

NE m/z 152E, NMN m/z 166MN m/z 180

NMN, MN m/z 149; NE, E m/z 135

E, NE m/z 107

NEH+ m/z 170; EH+ , NMNH+ m/z 184MNH+ m/z 198

E m/z 120; NE m/z 166

NMN m/z 134; MN m/z 148

Scheme 4. Decomposition of protonated catecholamines with X¼OH.

Copyright # 2006 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2006; 20: 1405–1421

DOI: 10.1002/rcm

Table 3. Transitions and neutral losses characterizing deprotonated or decationized catecholamines. In bold, transitions and

neutral losses used for the identification of compounds in the mixture

Compounds R4 R3 X Z Transitions Neutral losses DP� (V) CE�� (eV)

DOPAC[M–H]�m/z 167 H H H COOH 167! 123 44 (CO2) �18 �16HVA[M–H]�m/z 181 CH3 H H COOH 181! 137 44 (CO2) �36 �10HVA-d2

[M–H]�m/z 183 CH3 H H COOH 183! 139 44 (CO2) �36 �10VMA[M–H]�m/z 197 CH3 H OH COOH 197! 137 60 (CO2þCH4) �31 �30[MHPG–HþSO3K][M–K]�m/z 263 CH3 SO3K OH CH2OH 263! 165 98 (SO3þH2O) �35 �25



earlier.27 In the particular case of MN, a loss of the

methyl radical from [MH–H2O]þ is also observed.

The catecholamines analyzed in the negative ion mode

have the following structures:

R3O

R4OZ

X

R3 = H; SO3K

R4 = H; CH3

X = H; OH

Z = COOH; CH2OH

Few product ions are observed in the collision mass

spectra recorded in the negative ion mode; the compounds

are characterized with one transition only. The transitions

observed for the four catecholamines are tabulated in Table 3.

The most intense fragmentation of [DOPAC–H]� and

[HVA–H]� (X¼R3––H) corresponds to the loss of CO2, giving

m/z 123 and 137, respectively. In the case of [VMA–H]�

(X¼OH), the loss of CO2 is followed by a loss of methane

(R4H). The deprotonated HVA-d2, used as internal standard,

fragments as [HVA–H]� losing CO2. The decomposition of

these deprotonated compounds is summarized in Scheme 5.

Themechanism of fragmentation of these species is currently

being explored using theoretical calculations and the results

will be published later.

HO

R4O O

X

O

X=H, R4=H [DOPAC-H]- m/z 167

X=H, R4=CH3 [HVA-H]- m/z 181

X=OH, R4=CH3 [VMA-H]- m/z 197

[M-H]-

- CO

- R- C

Scheme 5. Decomposition of de


In the particular case of the MHPG sulfate salt, the

elimination of 98Da from [MHPG–HþSO3]� has been

attributed to the simultaneous losses of SO3 and H2O, as

proposed in Scheme 6.

IndolaminesThree indolamines, 5-HTP, 5-HT and HIAA, characterize the

metabolism of tryptophan (Trp). They have the following

structures:

R

W4'

NH

2'

W= H; OH

R=CH(NH2)(COOH); CH2NH2; COOH

The main fragmentations of these protonated compounds

are reported in Table 4.

TrpHþ and 5-HTPHþ are protonated aromatic a-amino

acids and fragment in the same way as TyrHþ and DL-

DOPAHþ (vide supra). However, at low energy, the

fragmentation involving loss of ammonia is prominent,

with the losses of [H2OþCO] being observed to a minor

extent. The loss of ammonia from Trp-d5 is accompanied by a

scrambling between the hydrogens of NH3, COOH and the

deuterium atoms in the 20 and 40 positions in the indole ring.

This exchange was described earlier by Lioe et al.31

HO

O

XR4

H

HO

O

O

H

2

4H

VMA m/z 137

DOPAC m/z 123HVA m/z 137

O2

protonated catecholamines.


DOI: 10.1002/rcm

Table 4. Transitions and neutral losses characterizing protonated indolamines. In bold, transitions and neutral losses used for the

identification of compounds in the mixture

Compounds R W Transitions Neutral losses DP� (V) CE�� (eV)

Trp CH(NH2)(COOH) H 205! 188 17 (NH3) 15 20MHþm/z 205 205! 159 46 (H2OþCO)

205! 146 59 (NH3þCH2CO) 15 25Trp-d5 CH(NH2)(COOH) H 210! 192 18 (NH2D) 31 15MHþm/z 2105-HTP CH(NH2)(COOH) OH 221! 204 17 (NH3) 20 10MHþm/z 221 221! 175 46 (H2OþCO)

204! 162 42 (CH2CO) 30 205-HT CH2NH2 OH 177! 160 17 (NH3) 20 21MHþm/z 177 160! 142 28 (CO)

177! 132 45 (NH3þCO) 20 35HIAA COOH OH 192! 146 46 (H2OþCO) 21 27MHþm/z 192


O

OH3CO O

HH

O2SO

O

CH3

O

O

- SO3 - H2O

[MHPG-H+SO3]-m/z 263 m/z 165

Scheme 6. Decomposition of [MHPG–HþSO3]�.


The protonated compounds substituted with R¼COOH

(HIAA) or NH2 (5-HT) lose [H2OþCO] and NH3, respect-

ively. The formation ofm/z 132 from 5-HTHþ corresponds to

successive losses of NH3 and 28Da.

GABA and metabolitesExcept for homocarnosine, the compounds involved in the

metabolic pathways of GABA have the following structures:

O

(CH2)n

OH

A

A= CH(NH2)(COOH); NH2; OH; COOH and n=2 or 3.

Homocarnosine is GABA esterified by histidine (His). The

lowest energy fragmentation of this protonated molecule

gives HisHþ. For the formation of HisHþ, we propose the

mechanism described in Scheme 7. This fragmentation

occurs from the amide-protonated form. The cleavage

between NHþ2 and CO is assisted by cyclization of the

leaving group. The ring formation and the transfer of

hydrogen from the amine function to the imidazole are

simultaneous.

The decomposition of HisHþ is well known.29,30 Succes-

sive losses of [H2OþCO] from HisHþ lead to the ion at

m/z 110.

GluHþ eliminates competitively29 H2O and [H2OþCO],

givingm/z 130 and 102, respectively. Successive eliminations

of [H2OþCO] from m/z 130 lead to m/z 84 (Scheme 8).


GABAHþ loses NH3 and H2O competitively. However, the

loss of NH3 predominates. [MH–NH3]þ competitively elim-

inatesCH2COor [H2OþCO] givingm/z 45 and 41, respectively

(Scheme 9). [MH–H2O]þ successively loses NH3 and 28Da,

leading to m/z 69 and 41, respectively. The fragmentation

mechanism of GABAHþ has been studied by means of

molecular orbital calculations and will be published later.

The loss of NH3 from protonated GABA-d6 does not

involve deuterium.

The compounds without an amine function (GHB, SA)

were analyzed in the negative ion mode. In both cases,

[M–H]� loses CO2 or a water molecule. In the case of

[GHB–H]�, a loss of H2 from [M–H–CO2]� is also observed.

PterinsThe studied pterins have the following structures:

HN

N NH2N

NOH

OCH2RHO

R = H; OH

Protonated pterins eliminate water. For both compounds,

this first loss of water is followed by a second loss of water or

a loss of 42Da. As the loss of 42Da is observed for both

pterins, the R group is not involved in this fragmentation.

The loss of 42Da is expected to correspond to NH2CN.

[NeoH–H2O]þ loses 30Da specifically. As this loss of 30Da

from [NeoH–H2O]þ is specific to NeoHþ, the R group is

considered to be involved in this fragmentation (Scheme 10).

Until now, the mechanisms of fragmentation of these

protonated compounds have been totally unknown.

Neutral losses (NL)Certain losses of small molecules (e.g. NH3, H2O,

[H2OþCO]) are often observed in the fragmentation of

protonated compounds of interest (Tables 2 and 4–6). These

neutral losses do not specifically characterize compounds.


DOI: 10.1002/rcm

NH2-CH(CH2)2COOH

H3N COOH

HO

O

GluH+

NHO

HO

H

O

HN

O

m/z 102

m/z 130

- H2O

m/z 84

m/z 148

- 2H2O - CO- H2O - CO

- H2O - CO

Scheme 8. Decomposition of GluHþ.

CH2

N

NH

COOHCH

O

NH2

NH

H

HCH+

CH2

N

NH

CH

NH2

CH2

NH

NH

COOHCH

NH2

NH

O

- H2O - CO

m/z 110

+

m/z 156

m/z 241

Scheme 7. Decomposition of HCHþ.

H3N

HO

O

GABAH+

[GABAH- NH3]+

[GABAH- H2O]+

- NH3

- H2O

m/z 45

m/z 41

- CH2CO

- H2O - CO

m/z 69m/z 104

m/z 87

m/z 86 - NH3

- H2O - NH3

m/z 41- 28 u

Scheme 9. Decomposition of GABAHþ.

[MH- 2H2O]+ HN

N NH2N

NO

O HCHO

MH+

BioH+ m/z 238; NeoH+ m/z 25

Bio m/z 202;Neo m/z 218

- 2H2O

R=H (Bio); OH (Neo)

Scheme 10. Decomposition



However, they give useful insight into functional groups

characterizing families of compounds.

In the positive ion mode and at low declustering potential,

losses of NH3 (NL17) andH2O (NL18) are associatedwith the

substitution of studied compounds by aliphatic amines and

hydroxyl functions, respectively. Losses of [H2OþCO]

(NL46) characterize amino acids or carboxylic acids. All

the collision mass spectra of the studied protonated

compounds include at least one of these transitions and

often two or three. At higher declustering potentials, losses of

H2O (NL18), CH3OH (NL32), [H2OþCO] (NL46) and

[CH3OHþCO] (NL60) characterize the substitution on the

aromatic ring of catecholamines.

Only six compounds were analyzed in the negative ion

mode. Neutral loss of CO2 (NL44) characterizes four

compounds substituted by a carboxylic function. Some

specific losses, such as NL60 and NL98, characterize VMA

and MHPG sulfate.

In a mixture, the search for neutral losses frequently

observed in the families of studied neurotransmitters (in

bold in Tables 2–6) will allow the detection of unexpected

metabolites. Then, these new compounds will be identified

from their collision mass spectra.

Analyses of reference mixture in acidic waterAs seen previously, the reference mixture of 28 compounds,

including 12 compounds of the tyrosine family, five of the

GABA family, four indolamines, two pterins and five

deuterated compounds used as internal standards, was

analyzed in the MRM and NL detection modes.

Multiple reaction monitoringExtracted chromatograms of the reference mixture are

presented in Figs. 3 (positive ion mode) and 4 (negative

ion mode). In these figures, each compound is characterized

by its most abundant MRM transition. Combining results

obtained in the positive and negative modes, all of the

28 compounds present in acidic water are observed but in

various abundances.

In the positive ionmode (Fig. 3), and at the beginning of the

chromatogram, several compounds are co-eluted: homo-

carnosine (RT¼ 2.0min), GABA (RT¼ 2.1min), glutamic

acid (RT¼ 2.2min) and NE (RT¼ 2.4min). This co-elution

does not affect the abundance of characteristic ions. The

coupling between LC and detection by MRM prevents

H

H2R

[MH- H2O]+- H2O

4

Bio m/z 220; Neo m/z 236

- H2O- NH2CN

[MH- H2O- NH2CN ]+

Bio m/z 178; Neo m/z 194

[MH- H2O- CH2O ]+

- H2O- CH2O

Neo m/z 206

of protonated pterins.


DOI: 10.1002/rcm

Table 5. Transitions and neutral losses characterizing ionized GABA metabolism compounds. In bold, transitions and neutral

losses used for the identification of compounds in the mixture

Compounds Structure Transitions Neutral losses DP� (V) CE�� (eV)

Positive modeHCMHþm/z 241

NH

N

CH2-CH

HNC

O

(CH3)3NH2

COOH

241! 156

156! 110

8546 (H2OþCO)

1545

1420

A n

Glu CH(NH2)COOH 2 148! 130 18 (H2O) 20 20MHþm/z 148 148! 102 46 (H2OþCO)

148! 84 64 (18þ46) 20 24GABA NH2 3 104! 87 17 (NH3) 20 14MHþm/z 104 104! 86 18(H2O)

104! 69 35 (H2OþNH3) 20 19104! 45 59 (NH3þ42)87! 41 46 (H2OþCO)

GABA-d6 NH2 3 110! 93 17 (NH3) 26 17MHþm/z 110

Negative modeA n Transitions Neutral losses DP (V) CE (eV)

GHB 103! 85 18 (H2O) �24 �14[M–H]�m/z 103 OH 3 103! 57 46 (CO2þH2) �24 �20SA 117! 99 18 (H2O) �26 �14[M–H]�m/z 117 COOH 2 117! 73 44 (CO2) �26 �17


Table 6. Transitions and neutral losses characterizing of protonated pterins. In bold, transitions and neutral loses used for the

identification of compounds in mixture

Compounds R Transitions Neutral losses DP� (V) CE�� (eV)

Bio H 238! 220 18 (H2O) 40 24MHþm/z 238 238! 202 36 (2 H2O)

238! 178 60 (18þ42) 40 28Neo OH 254! 236 18 (H2O) 49 20MHþm/z 254 254! 218 36 (2 H2O)

254! 206 48 (18þ30) 49 25254! 194 60(18þ42)



spectral suppression. The limits of detection (LODs) of the

studied compounds depend on their polarity and are found

to be between 10�8 and 10�10M in the positive ion mode and

between 10�7 and 10�10M in the negative ion mode. These

LODs are sufficient for the detection of the greatest part of the

compounds of interest present in biological fluids.32 A low

mass resolution on Q1 and Q3 reduces these LODs (by about

a factor of 4).

Neutral lossesBy combining the RT and at least one of the following neutral

losses: NH3 (NL17), H2O (NL18), MeOH (NL32) and

[H2OþCO] (NL46), all the basic compounds included in

the mixture are detected and identified.

In the negative ion mode, the classical loss of CO2 (NL44)

leads to the detection of SA, HVA and DOPAC. This loss,


associated with methane elimination (NL60), identifies

VMA. For the detection of MHPG sulfate salt, a specific

loss is selected (NL98). All the acidic compounds present in a

mixture, except GHB, are detected using these three neutral

losses.

However, the ion current associated with the detection of

each compound is lower using NL than when using the

MRMmode. A scanned detectionmode such as theNLmode

is less sensitive than a reaction monitoring mode (MRM).

Analyses of biological fluids spiked with theneurotransmitter mixture

Matrix and resolution effectsThe matrix effect induced by biological fluids has been

evaluated, in the MRM mode, with internal standards. The


DOI: 10.1002/rcm


abundances of characteristic transitions detected from the

reference solution and from various spiked biological fluids

(CSFS, UrineS and AFS) have been compared. These

abundances are given in Table 7 as well as the retention

times of the compounds of interest.

For all standards, the retention times increase by several

tenths of a minute in biological fluids. A relatively small

decrease in the abundance of ions (a factor of 2 to 10) is

observed for all deuterated compounds analyzed in

biological fluids, except for GABA-d6. In the case of UrineS

andAFS, the abundances of ions characterizing GABA-d6 are

reduced by a factor of 100. As a salt effect was suspected, the

reference mixture was diluted in physiological serum. The

results acquired by MRM are presented in Table 8. All

internal standards are detected at the same retention times in

physiological serum and in various biological fluids,

exhibiting a small salt effect on retention times.

Except for GABA-d6, the physiological serum has little

influence on the abundances of detected ions. Moreover, the

1 2 3 4 5 6 7 8

4.0e4

5.8

1 2 3 4 5 6 7 8

1.4e56.0

1 2 3 4 5 6 7 8

1.00e55.0

1 2 3 4 5 6 7 8

7.5e54.9

.

1 2 3 4 5 6 7 8

1.4e63.7

1 2 3 4 5 6 7 8

8.0e52.4

1 2 3 4 5 6 7 8

5.9e52.2

1 2 3 4 5 6 7 8

4.0e5

2.1

1 2 3 4 5 6 7 8

3.8e52.1

1 2 3 4 5 6 7 8

1.00e52.0

spc ,

ytisn

etnI

spc ,

ytisn

etnI

spc ,

ytisn

etn I

spc ,

ytis n

etn I

spc ,

ytisn

etnI

s pc ,

y tisn

e tnI

s pc ,

yt isn

etnI

s pc ,

ytisn

etnI

spc ,

ytis n

etnI

spc ,

ytisn

etnI

Figure 3. Extracted chromatograms of 21 compounds dissolved a

mode.


decrease in the abundance of ions observed for GABA-d6 is

weaker in physiological serum than in biological fluids. In

conclusion, a matrix effect, depending on the internal

standard and the biological fluid, is observed. The salt and

matrix effects are prominent at the beginning of the

chromatogram only (Table 8). A small matrix effect is

observed for HVA-d2 in the negative ion mode.

The sensitivity of detection by MRM increases on

decreasing the mass resolution on Q1 and Q3 (vide supra).

This is illustrated in Table 9, with the abundances of MRM

transitions characterizing internal standards present in

spiked biological fluids, at low and unit mass resolution.

Detection of neurotransmitters in spiked biological fluidsThe 23 studied compounds were searched for in spiked

biological fluids, namely CSFS, UrineS and AFS, using MRM

and NL as detection modes. The results are reported in

Tables 10(a) and 10(b) for the positive and the negative ion

modes, respectively. Whatever the biological fluid, all the

9 10 11 12 13 14 15 16

9 10 11 12 13 14 15 16

9 10 11 12 13 14 15 16

9 10 11 12 13 14 15 16

9 10 11 12 13 14 15 16

NEm/z 170 → 152

9 10 11 12 13 14 15 16

9 10 11 12 13 14 15 16

9 10 11 12 13 14 15 16

9 10 11 12 13 14 15 16

HCm/z 241 → 156

9 10 11 12 13 14 15 16

Gaba-d6m/z 110 → 93

Glum/z 148 → 84

Gabam/z 104 → 87

E, NMNm/z 184 → 166

DAm/z 154 → 137

Neom/z 254 → 206

DL-DOPAm/z 198 → 152

DL-DOPA-d3m/z 201 → 154

Time, min

Time, min

Time, min

Time, min

Time, min

Time, min

Time, min

Time, min

Time, min

Time, min

t 10�6 M in acidic water and analyzed by MRM in the positive


DOI: 10.1002/rcm

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

2.7e5

10.4

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

5.9e58.3

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

9.0e56.8

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

3.3e58.1

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

8.6e512.3

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

1.2e612.4

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

6.6e511.2

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

1.22e611.0

1.3e5

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

14.6

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

3.4e6 10.8

5-HTm/z 177 → 160

5-HTPm/z 221 → 204

3-MTm/z 168 → 151

Biom/z 168 → 151

Tyr-d4m/z 186 → 169

Tyrm/z 182 → 165

MNm/z 198→180

TRPm/z 205 → 188

TRP-d5m/z 210 → 192

HIAAm/z 192 → 146

Time, min

Time, min

Time, min

Time, min

Time, min

Time, min

Time, min

Time, min

Time, min

spc ,

ytisn

etnI

spc ,

ytisn

etnI

spc ,

ytisn

etn I

spc ,

ytisn

etnI

spc ,

ytisn

etnI

spc ,

ytisn

etnI

spc ,

ytisn

etnI

spc ,

yt isn

etn I

spc ,

ytisn

etnI

spc ,

ytisn

etnI

Figure 3. (Continued)


compounds spiked into the biological fluid are observed

usingMRM transitions. All the compounds spiked into CSFS

and UrineS are also observed in NL mode. However, some

neurotransmitters included inAFS are not observed: (i) in the

positive ion mode, NE and 5-HT; (ii) in the negative ion

mode, MHPG sulfate and DOPAC. The detection of these

compounds from AFS may be disturbed by the presence in

this biological fluid of intrusive compounds (matrix effect).

Analyses of crude biological fluids

Detection of neurotransmitters in crudebiological fluidsFinally, the MRM and NL acquisition modes were used for

the detection of compounds of interest in crude biological

fluids. The results are tabulated in Table 11. Using the MRM


detectionmode, most of the studied compounds are detected

in biological fluids, except for NMN, DA andMN in CSF and

MN in AF. As the NL acquisition mode is less sensitive than

the MRM mode, some of the compounds of interest are not

detected, in agreement with their low concentration in

biological matrices.

Interest in neutral lossThe NL acquisition mode is used for the detection of

compounds not included in the reference solution. Intrusive

compounds are detected byNL by comparing corresponding

chromatograms of the reference mixture and of crude

matrices. Depending on the selected NL, new peaks appear

in the chromatograms of crude biological matrices.

As a first example, the chromatograms recorded from the

reference solution and fromCSF usingNL46 are compared in


DOI: 10.1002/rcm

Table 7. Retention times (RT) and abundances (counts/second (cps)) of ions associated with characteristic transitions of internal

standards detected by MRM in the reference solution and in various spiked biological fluids

Internal standards RT (min) Reference RT (min) CSFS UrineS AFS

GABA-d6 2.1 2� 106 2.4 8� 104 4.2� 104 4� 104

110! 139DL-DOPA-d3 5.8 5� 105 6 1.4� 105 4.6� 104 9� 104

201! 154Tyr-d4 8.1 4.5� 106 8.2 4.5� 105 2� 104 1.9� 105

186! 169Trp-d5 12.3 1.2� 106 12.3 1.1� 106 1.5� 105 7.4� 105

210! 192HVA-d2 14.3 5.5� 04 14.3 3.5� 104 2.3� 104 1.8� 104

183! 139

Table 8. Retention times (RT) and abundances (cps) of ions associated with characteristic transitions of internal standards

detected by MRM in the reference solution and in physiological serum

Internal standards RT (min) Reference RT (min) Physiological serum

GABA-d6 2.1 2� 106 2.4 2.5� 105

110! 139DL-DOPA-d3 5.8 5� 105 6 2.9� 105

201! 154Tyr-d4 8.1 4.5� 106 8.2 4.4� 106

186! 169Trp-d5 12.3 1.2� 106 12.3 1.0� 106

210! 192HVA-d2 14.3 5.5� 104 14.3 2� 104

183! 139

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

1.8e610.1

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

3.1e510.0

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

2.0e55.5

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

4.0e45.5

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

2.0e514.2

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

9.0e512.0

GHBm/z 103 → 57

SAm/z 117 → 73

VMAm/z 197 → 137

MHPG sulfatem/z 263 → 150

DOPACm/z 167 → 123

HVAm/z 181 → 137

spc ,

ytisn

etn I

s pc ,

y tis n

etnI

spc ,

ytisn

etnI

spc ,

ytisn

etnI

s pc ,

y tisn

etnI

spc ,

y tis n

e tnI

Time, min

Time, min

Time, min

Time, min

Time, min

Time, min

Figure 4. Extracted chromatograms of seven compounds dissolved at 10�5 M in acidic

water and analyzed by MRM in the negative mode.


DOI: 10.1002/rcm


Table 10. (a) Presence (þ) or absence (�) of reference compounds detected in the positive ion mode, in spiked biological fluids,

using RT, MRM and/or NL, Q1 and Q3 working at unit mass resolution. (b) Presence (þ) or absence (�) of reference compounds

detected in the negative ion mode, in spiked biological fluids, using RT, MRM and/or NL, Q1 and Q3 working at unit mass resolution

Spiked biological fluids! CSFS UrineS AFS

Compounds RT (min) MRM NL MRM NL MRM NL

(a)GABA 2.2 þ þ(17; 18; 46) þ þ(18; 46) þ þ(18; 46)HC 2.3 þ þ(46) þ þ(46) þ þ(46)Glu 2.3 þ þ(18; 46) þ þ(18; 46) þ þ(18; 46)NE 2.6 þ þ (18) þ þ(18) þ �E 3.6 þ þ(46) þ þ(18; 46) þ þ(18)NMN 3.8 þ þ(17; 18; 32) þ þ(18; 32) þ þ(17; 18; 32)DA 5.2 þ þ(17) þ þ(17) þ þ(17)Neo 5.2 þ þ(18) þ þ(18) þ þ(18)DL-DOPA 6.1 þ þ (17, 46) þ þ(46) þ þ(46)MN 6.9 þ þ(18; 32) þ þ(18) þ þ(18)Tyr 8.0 þ þ(17;18;46) þ þ(17; 46) þ þ(17; 46)Bio 10.1 þ þ(18) þ þ(18) þ þ(18)3-MT 10.8 þ þ(17;32) þ þ(17, 32) þ þ(17; 32)5-HT 11 þ þ(17) þ þ(17) þ �5-HTP 11.3 þ þ(17; 18) þ þ(17; 46) þ þ(17; 46)Trp 12.5 þ þ(17;18; 46) þ þ(17; 46) þ þ(17;46)HIAA 14.8 þ þ(46) þ þ(46) þ þ(46)

(b)GHB 5 þ þ(44) þ (44) þ þ(44)SA 5.6 þ þ (44) þ þ (44) þ þ(44)VMA 10.5 þ þ(60) þ þ(60) þ þ(60)MHPG sulfate 11 þ þ(98) þ þ(98) þ �DOPAC 12.1 þ þ(44) þ þ(44) þ �HVA 14.2 þ þ(44) þ þ(44) þ þ(44)

Table 9. Abundances (cps) of ions associated with characteristic transitions of internal standards detected by MRM in the

reference solution and in various spiked biological fluids, Q1 and Q3 working either at unit or low mass resolution (R)

Spiked biological fluids! CSFS UrineS AFS

Internal standards Unit R Low R Unit R Low R Unit R Low R

GABA-d6 8� 104 2.7� 105 4.2� 104 2� 105 4� 104 1.5� 105

110! 139DL-DOPA-d3 1.4� 105 106 4.6� 104 3� 105 9� 104 6� 105

201! 154Tyr-d4 4.5� 105 3.9� 106 2� 104 3� 105 1.9� 105 2.5� 106

186! 169Trp-d5 1.3� 106 6� 106 1.5� 105 9.5� 105 7.4� 105 3.8� 106

210! 192HVA-d2 3.5� 104 1.19� 105 2.3� 104 1.4� 105 1.8� 104 105

183! 139


Fig. 5. The chromatogram of CSF exhibits some new peaks,

among them a peak at RT¼ 11.4min. At this RT, the ion

precursor ofNL46 is detected atm/z 166. This ion is suspected

to be protonated phenylalanine, PheHþ, the precursor of

tyrosine. The fragmentation of protonated aromatic amino

acids is well known (vide supra): they lose [H2OþCO] andNH3

competitively, then [MH–46]þ loses NH3.

The presence of Phe may be confirmed by recording the

chromatogram of CSF associated with NL17. The latter also

exhibits a peak at RT¼ 11.4min (not observed in the


corresponding chromatogram of the reference solution).

Two ions, precursors of this loss, are observed atm/z 166 and

120, identified as PheHþ and [PheH–H2O–CO]þ, and confirm

the presence of phenylalanine in CSF (Fig. 6).

3-O-Methyl DOPA (3-OMD) is the 3-OH-methylated

metabolite of DL-DOPA. The detection of this compound

in urine, not included in our reference mixture, is a second

example. As 3-OMDHþ is a protonated aromatic amino acid

like DL-DOPAHþ, competitive and successive losses of

[H2OþCO] and NH3 are expected.


DOI: 10.1002/rcm

282624222018161412108642Time, min

0.05.0e61.0e71.5e72.0e72.5e7

3.0e73.5e7

Inte

nsity

, cps

8.8

5.9

mixture(1) NL 46 on Reference

5.4

282624222018161412108642Time, min

0.02.0e64.0e66.0e68.0e61.0e71.2e71.4e7

Inte

nsity

, cps

11.413.60

(2) NL 46 on CSF

166

m/z

Figure 5. Chromatogram of NL46 on (1) reference mixture and on (2) CSF; insert:

precursor ions of NL46 at RT¼ 11.4 min.

Table 11. Presence (þ) or absence (�) of reference compounds detected in crude biological fluids using RT, MRM and/or NL, Q1

and Q3 working at unit mass resolution

Biological fluids! CSF Urine AF

Compounds RT (min) MRM NL MRM NL MRM NL

GABA 2.2 þ þ(18; 46) þ þ(46) þ þ(46HC 2.3 þ þ(46) þ þ(46) þ þ(46)Glu 2.3 þ þ(46) þ þ(18; 46) þ þ(18; 46)NE 2.6 þ � þ þ(18) þ �E 3.6 þ � þ � þ �NMN 3.8 � � þ þ(17; 18; 32) þ �DA 5.2 � � þ þ(17) þ �Neo 5.2 þ þ(18) þ þ(18) þ þDL-DOPA 6.1 þ � þ � þ �MN 6.9 � � þ � � �Tyr 8.0 þ þ(17; 18; 46) þ þ(17; 46) þ þ(17; 46)Bio 10.1 þ þ(18) þ þ(18) þ þ3-MT 10.8 þ � þ þ(17; 32) þ �5-HT 11 þ � þ � þ �5-HTP 11.3 þ � þ þ(46) þ þ(46)Trp 12.5 þ þ(17; 46) þ þ(17; 46) þ þ(17; 46)HIAA 14.8 þ þ(18; 46) þ þ(46) þ þ(46)GHB 5 þ þ(44) þ � þ þ(44)SA 5.6 þ þ (44) þ � þ þ(44)VMA 10.5 þ þ(60) þ þ(60) þ þ(60)MHPG sulfate 11.0 þ � þ þ(98) þ �DOPAC 12.1 þ þ(44) þ þ(44) þ �HVA 14.2 þ þ(44) þ þ(44) þ þ(44)


The precursor ions at m/z 212 and 195, expected to be

[3-OMDH]þand [3-OMDH–NH3]þ, respectively, were sea-

rched for in the chromatogram of urine associated with NL46

and correspond to a peak detected at RT¼ 11.4min. In the

samemanner, precursor ions atm/z 212 and 166, correspond-


ing to [3-OMDH]þ and [3-OMDH–H2O–CO]þ, were

searched for in the chromatogram of urine associated with

NL17 and also correspond to a peak detected at

RT¼ 11.4min (Fig. 7). These results are in agreement with

the presence of 3-OMD in the urine.


DOI: 10.1002/rcm

2322212019181716151413121110987654321 24 25

1.0e62.0e63.0e64.0e65.0e66.0e67.0e68.0e69.0e6

Inte

nsity

, cps

300280260240220200180160140120100806040m/z

5.0e41.0e51.5e52.0e52.5e53.0e5

120

166In

tens

ity, c

ps

Time (min)

RT= 11.4 minions at(2) Precursor

(1) NL 17 on CSF11.4

13.2

9.7

Figure 6. (1) Chromatogram of NL17 on CSF, (2) precursor ions at

RT¼ 11.4 min.

282624222018161412108642

Time, min

Inte

nsity

, cps

11.4

2.25.7 12.18.0

14.1

282624222018161412108642Time, min

Inte

nsity

, cps

13.9

282624222018161412108642

(a)

0.02.0e74.0e76.0e7

0.0

5000.0

1.0e4

1.4e4

0.01.0e5

2.0e5

3.0e5

Inte

nsity

, cps

11.4

Time, min

(1) NL 46 on Urine

(2) Extracted ion current at m/z 195


11.4

2.2

282624222018161412108642Time, min

0.01.0e72.0e73.0e74.0e74.8e7

Inte

nsity

, cps

13.2

12.113.87.12.4

(b)

282624222018161412108642Time, min

0.01.0e52.0e53.0e54.0e55.0e5

Inte

nsity

, cps

7.1 12.4

10.811.4

282624222018161412108642Time, min

0.0

5.0e4

1.0e5

1.5e5

Inte

nsity

, cps

11.4

(1) NL 17 on Urine



Figure 7. (a) 1, Chromatogram of NL 46 on urine; 2, extracted ion current at m/z 195;

3, extracted ion current at m/z 212. (b) 1, Chromatogram of NL 17 on urine; 2,

extracted ion current at m/z 166; 3, extracted ion current at m/z 212.


Interest in neurotransmitter profilesin MRM and NLThe interest of this study is highlighted in an example

comparing the profiles of normal and pathological (Neuro-


blastom) urine samples. The urine samples were obtained

from patients of the same age. For this comparison, the

concentration of creatinine was normalized. Partial and

extracted MRM profiles recorded in the positive ion mode


DOI: 10.1002/rcm

DAm/z 154 137

DAm/z 154 91

NEm/z 170 152

C) Extracted chromatogram for NMN transitions

Time, min

spc ,

ytisn

etnI

spc ,

ytis n

etnI

spc ,

ytisn

etnI

spc ,

ytisn

etn I

spc ,

y tis n

etnI

spc ,

ytisn

e tnI

2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.Time, min

5.00e5

1.27e65.3

4.2

Time, min

2.0e5

4.0e5

6.0e55.3

Time, min

2.0e53.0e5

4.5e55.3

Time, min

2.0e5

4.0e5

4.2

Time, min

2.0e4

4.0e4

6.0e47.3e4

4.2

1.0e4

2.0e4

3.0e4

2.7

NMNm/z 184 166

NMNm/z 166 149

2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.

2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.

2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.

2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.

2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.

NE

NMN

DAA) Partial chromatogram in MRM mode

B) Extracted chromatogram for DA transitions

D) Extracted chromatogram for NE transition

6.0e5

→

→

→

→

→

Figure 8. Partial (A) and extracted (B–D) chromatograms showing the difference between a

control urine (bold line) and a urine from a patient affected by a neuroblastom (dotted line)

analyzed by MRM in positive ion mode.

1 2 3 4 5 6 7 8 9 10 11 12 13Time, min

5.0e5

1.0e6

1.5e6

2.0e6

2.5e6

ytisn

etnI

s pc ,

11.23

MHPG sulfatem/z 263 → 165

12.0 13.0 14.0 15.0 16.0 17.0

Time, min

2.0e4

6.0e4

1.0e5

1.4e5

1.8e5

spc ,

ytisn

e tnI

14.2

HVAm/z 181 → 137

A) Extracted chromatogram for MHPG sulfate transition

B) Extracted chromatogram for HVA transition

Figure 9. Extracted (A, B) chromatograms showing the difference between a

control urine (bold line) and a urine from patient affected by a neuroblastom

(dotted line) analyzed by MRM in negative ion mode.


DOI: 10.1002/rcm


8 9 10 11 12 13 14 15 16 17Time, min

ytisn

etnI

spc ,

1.00e6

2.00e6

3.00e6

4.00e6

5.00e6

6.00e6

7.00e6

8.00e6

9.00e6

1.00e7

1.10e7

12.8

9.816.6

m/z 233

m/z 263 MHPG sulfate

m/z 249

Figure 10. Partial chromatograms of a control urine (bold

line) and a urine from a patient affected by a neuroblastom

(dotted line) analyzed by NL98 in negative ion mode.


are presented in Fig. 8. Extracted MRM and partial NL98

profiles acquired in the negative ion mode are shown in

Figs. 9 and 10, respectively.

The overlay of both partial profiles in Fig. 8(A) shows, for

the pathological urine, a weak increase of NE abundance and

a large increase of DA and NMN abundances. Figures 8(B)–

8(D) present the extracted chromatograms associated with

each transition characterizing these neurotransmitters.

Figure 9 exhibits a large increase in MHPG sulfate and

HVA abundances. These anomalies detected in Figs. 8 and 9

were expected for this disorder.

In Fig. 10, the superposition of profiles exhibits a large

increase in the abundance of the peaks corresponding to

m/z 263 and 233 and the appearance of a peak corresponding

to m/z 249. These ions are associated with a search for a

neutral loss of 98Da (SO3þH2O). The ion at m/z 263 is

identified as theMHPG sulfate. As the ions atm/z 233 and 249

also contain the sulfate function and the hydroxyl group in

the benzylic position, they are considered as metabolites of

MHPG sulfate. According to their retention times, the

metabolite at m/z 249 is expected to include R4¼H and

the one at m/z 233, Z¼CH3.

CONCLUSIONS

In this work, a LC/ESI-MS/MS method, using multiple

reaction monitoring (MRM) and neutral loss (NL) as

acquisition modes, was developed for the screening and

qualitative determination of 23metabolites related to three of

the most important families of neurotransmitters in bio-

logical fluids. This method, easy to use, requires no

extraction procedure of compounds in amixture in biological

fluids and requires only small amounts of biological fluids.

It has been shown in the reference mixture that the two

acquisition modes (MRM and NL) are complementary. The

MRM acquisition mode gives a qualitative profile of


neurotransmitters which is specific for certain neurotrans-

mitter disorders and will be useful to compare the tissue-

specific enzymatic content for physiological purposes. It will

be a method of choice for quantifying, in biological fluids, if

necessary, neurotransmitters and metabolites identified with

the qualitative profiles. The NL acquisition mode gives

information about unexpected metabolites present in

biological matrices.

The comparison of reference profiles recorded by MRM

and NL modes with profiles of children showing specific

neurological signs recorded with the same conditions will

allow the rapid diagnosis of given diseases and help the

understanding of metabolic error(s).

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DOI: 10.1002/rcm

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