Pre-treatment and one-shot separating analysis of

whole catecholamme metabolites in plasma by usingH

LC/MS

ToMOMI HASEGAWA, KASTUYA WADA, Eiso HIYAMA, AND TsuTOMU MASUJIMA*

Graduate School ofBiomedical Science, Hiroshima University, 1-2-3 Kasumi, Minami-ku,

Hiroshima, Hiroshima, 732-8551, Japan.

+8 1-(0)82-257-5300

+8 1-(0)82-257-5304

tsutomu@n irosnima-u. ac.jpThis work was partly supported by Gr肌t血-Aids for Scientific Research舟om the Ministry of

Education, Science, Sports, and Culture of Japan.

Background: Catecholamines are biogenic amines playing an important role in the nervous system. Part of

catecholammes have been used as tumor makers of phenochromocytoma, paraganglioma and neuroblastoma.

The analysis of total catecholamine metabolites should be useful for one shot screening of multi aspects of

diseases. However it is difficult to conduct, because the catecholamine metabolites are divided into three

groups:丘ve amines, one ammo acid and three carbonic acids.

Method: Catecholamines and small molecules were separated from plasma proteins by an internal surface

reversed phase column (protein-coated ODS column) a去d analyzed by LC伽S using ESI-TOF,伽S.

Result: Using a reversed phase column and hydrophilic mobile phases, we succeeded in the separation of nine

catecholamin.es, all of which structures are similar. These nine substances were eluted in the following order:

Norepinephrme, Epinephrine, Normetanephrine, Dopamine, Metanephrine, DOPA, VMA, DOPAC and HVA.

The reproducibility of this method was acceptable. The highest C.V. was 7.4%. In addition, various types of

compounds were separated from and detected in plasma proteins by applying LC/MS.

Conclusion: Plasma direct injection method, which uses an internal surface reversed phase column and ion-pair

reagent, allowed us to separate small molecules from plasma proteins. MS detected some compounds that HPLC

could not succeed in the separating and detecting with UV detection. We think that the established method can

be applied to find new markers in neuroblastoma, comparing the plasma of patients to that of normal infants.

The method can be also used to help for making a diagnosis of other diseases and finding their new makers.

Keywords: neuroblastoma, Catecholamine, Internal surface reversedphase column , LG/MS

DOPA, 3.4-Dihydroxy-phenylalanine; DOPAC, 3. 4-Dihydroxy-phenylacetic acid; HVA,

Homovamllic acid; VMA, Vanillomandelic acid; and ESI-

TOF/MS, electrospray ionization-time of flight/mass

sp ectrometory.

Introduction

Neuroblastoma is a malignant tumor especially found in infants. The frequency of the

incidence is the second highest after that of leukemia. Because of its high frequency and the

possibility of curing the disease with early detection, the nation-wide neonatal mass-screening

at six months of age was conducted in Japan between 1985 and 2004 in Japan. Infants with

Neuroblastoma produce an excess of catecholamines compared to normal infants (1).

Therefore, a considerable amount of VMA and HVA, the final metabolites of catecholamines,

are excreted into the urine (2, 3, 4, 5, 6, 7). The present mass-screening observed an increase

of the final metabolites by肝LC (8, 9). However this screening-method had problems with

both accuracy of metabolite measurement and the determination of a definitive cut-off value.

The committee of Ministry of Health, Labour and Welfare in Japan reached the conclusion

that the screening has no effect on mortality from Neuroblastoma, and thus it was suspended

(10, 1 1). Contrary to the negative opinions, some believe that the mass-screening actually

helped reduce mortality due to Neuroblastoma. This disease is still a convalescent

unsatisfactory disease and therefore, the mass-screening is vital to a reduction of the mortality

due to infantile cancers (12, 13, 14). In order to restart the mass-screening, the current method,

which targets only the final substances (VMA and HVA) and considers metabolic substances

of catecholamine to be an insignificant subject, needed to be reexamined. We analyzed all

metabolic substances of catecholamine, including metabolic by-products, and set up an

analytical method using LC伽S. The urine is a proper sample for the screening, posing the

least burden and risk to infants and their parents. However, the urine does not contain all of

the compounds of the human body and also has a problem with the stability of components

generated by contamination. The blood, on the other hand, contains all of the compounds of

the human body. Thus we established a test method for analysis of the plasma.

In the plasma, small molecules unite with plasma proteins especially albumin. For the

analysis of small molecules in the plasma, these small molecules needed to be separated from

plasma proteins. For the separation, we employed a direct plasma injection analysis, which

uses an血ernal su血ce reversed phase column. Fig. 1 shows the characteristics of the

column. The outside surface of porous resins was coated with BSA, so that the column did not

adsorb for the plasma proteins, but still had the reverse-phase characteristics for small

molecules. The internal surface reversed phase column was selected as a precolumn for the

deproteinization and a trapping of small molecules(1 5, 16).

F IG . 1

In the human body, catecholamines are formed by the following sequence of reactions:

Tyro sme-サ3. 4-Dihydroxy-phenylalanine (D OPA)-> D op amine->Norepinephrine->

Epmephrine. They have physiological activity respectively. A洗er they performed their roles,

DOPA is metabolized and becomes Homovanillic acid (HVA) via 3.4-Dihydroxy-

phenylaacetic acid (DOPAC). Dopamine also changes into HVA. Norepinephrine transforms

into Normetanephrine, and Epinephrine becomes Metanephrine. They then both transforms

into Vanillomandelic acid (VM勾These reactions are catalyzed by the following enzymes:

( l )Tyro sine-hydroxylase, (2)D OPA-decarboxylase, (3 )Dopamine- p -hydroxylase5

(4)Phenylethanolamine-N-methyltransferase, (5 ) catechol-O-methyl-tranceferase,

(6)monoaminかoxidase. Finally HVA and VMA are excreted to the urine (Fig. 2).

Materials and Methods

C血emi c al s

DOPA, Dopamine, DOPAC, Epinephrine, Normetanephrine, Metanephrine, HVA, VMA and

Formic acid were obtained from SIGMA. Norepinephrine was from Fluka. Methanol,

acetomtrile, Ammonia solution, Ammonium Hydrogencarbonate and､.

pentadecafluorooctanoic acid (CF3(CF2)6COOH) were purchased from KANTO CHEMICAL.

Trifluoroacetic acid (CF3COOH), Pentafluoropropionic acid (CF3CF2COOH),

Heptafluorobutyric acid (CF3(CF2)2COOH) were obtained from ALDRICH.

Nonafluorovaleric acid (CF3(CF2)3COOH) was from TOKYO KASEI KOGYO. Methanol

and acetonitrile were肝LC grade, and all other reagents were analytical grade. Water was

dei｡nized and purifed by Milli-QR Academic AI 0 purification system from Mllipore.

Sample preparation

Standard solution of catecholamine metabolites was dissolved in water at a concentration of 1

mg/mL and mixed all in one solution to a final concentration of 100 ng/mL. The mixed

standard solution was diluted by foetal bovine serum or human plasma to be 10 ng/mL and

used as samplかspiked plasma.

In strument ati o n

The HPLC-UV system was Gulliver Series from Jasco, consisting ofDG-980-50 3-Line-

Degasser, LG- 1 580-02 Termary Gradient Unit, PU-980 Intelligent肝LC Pump, and UV-970

Intelligent UV/VIS Detector. Pretreatment was performed by using the internal surface

reversed phase column (TSK precolumn BSA-ODS:TOSOH) and a column packed anion

exchange resin (TOYOPEARL QAE-550:TOSOH). The analysis column was L-column

ODS (150x4.6 mm, 5 |iim) from CERI. MS analysis was performed using a Mariner�"ESI-

TOF/MS舟om Applied Biosystems.

Chromatographic Conditions

Mobile phases for the pretreatment column were (A) water containing 1 % offormic acid

and (B) 200 mM Ammonium Hydrogencarbonate buffer/acetonitrile (5 :95 v/v). The internal

surface reversed phase column was equilibrated by solvent (A) at a flow rate of 0.5 mL/min,

and samples were injected into the HPLC system. Following the removal of plasma proteins

and other hydrophilic compounds, the mobile phase was changed to solvent (B) by switching

a six-port valve. Catecholamine metabolites and hydrophobic compounds were eluted, and

the fractions were collected. Then the fractions were evaporated. The residue was dissolved

in lOOいL of mobile phase, and 20 |oL was injected into the LCIMS system. Two mobile

phases used for the analysis of these treated samples were (A) water containing 1 % of

formic acid, and (C) methanol containing 1 % offormic acid. Separation was conducted at a

flow rate of 0.2 mL/min. For the separation of amines, the concentration of solvent (C) was

5% for the first 18 minutes. In order to elute VMA, DOPAC and HVA the concentration of

solvent (C) was raised to 45% for the next 12 minutes. The UV detector was set to monitor

280nm. The MS was used in the positive ion ESI mode.

Results and Discussion

S eparation of catecholamine metabolites

We developed a method for the HPLC separation of catecholamine metabolites. The

catecholamine metabolites were divided into two groups: five amines and one amino acid, and

three carbonic acids. In order to separate five amines and one ammo acid, of which structures

are si血Iar, the hydrophilic mobile phase needed to be used. In this case, we used l% of

formic acid/methanol (5:95 v/v) and accomplished a good separation. Fig. 3 shows the LC

and MS chromatograms. The elution order was Norepinephrine, Epinephrine,

Normetanephrme, Dopamine, Metanephrine and DOPA. The peaks ofNorepinephrine were

m/z! 52 ([M+H-H2O] ) and m/z!70 ([M+H]+). Epinephrine was detected nVz! 84 ([M+H]+),

Normetanephrine was detected m/z!66 ([M+H-H2O】 ) and m/z!84 ([M+Hf), Dopamine was

detected m/z! 54 ([M+H】 ), Metanephrine and DOPA were detected m/z! 80 (【M+H-H2O】+)

and m/z!98 ([M+H]+).

DOPAC, HVA and VMA have a strong affinity with ODS. To accelerate the elution of these

three substances, the concentration of the organic mobile phase was raised from 5 to 45 %.

As a result, all procedures were carried out within 60 minutes. The elution order of carbonic

acids was VMA, DOPAC and HVA (Fig. 4). The peaks ofDOPAC were m/z!69 ([M+H]+),

m/z!86 ([M珊HJ ), and m/z!91 ([M+Na]+). HVA was detected m/z!83 ([M+H】-*-), m/z200

([M+NHU] ), and m/z205 ([M+Naf). No peak was found in VMA. It is suspected that

because V加仏had a hydroxyl group close to the carboxyl group, the pKa was lower

compared to other carbonic acids. Thus VMA could not ionize well with the ESI positive

mode.

By trapping of catecholamine metabolites to the internal surface reversed phase column,

we separated catechola血ne metabolites and small molecules鮎m plasma proteins. Plasma

proteins ran through the column without being trapped, because of the size exclusion and the

coated BSA. We set the elution time ofproteins by HPLC-UV and Lowry method

(absorbance of700 nm). At a flow rate ofO.5 mL/min and 100 jjL of sample volume, plasma

proteins were eluted fro血the column within three minutes (Fig. 4-a,b). Under these

conditions the internal surface ODS did not hold catecholamine metabolites enough, because

they were ionized. In order to increase the affinity of ODS, we used an volatile ion-pair

reagent(1 7, 1 8). With the reagent, Trifluoroacetic acid (CF3COOH) ,Pentafluoropropionic acid

(CF3CF2COOH), Heptafluorobutyric acid (CF3(CF2)2COOH) , Nonafluorovaleric acid

(cF3(CF2)3COOH) arid pentadecafluorooctanoic acid (CF3(CF2)6COOH) were tested ( Fig.4-

c). We accomplished a good separation using 0. 1 mM pentadec弧Iorooctanoic acid/ 1 %

formic acid (Fig. 4-d). With this mobile phase, ion-pair reagent was not removed by

evaporation. We placed a column packed strong anion exchange resin between the switching

six-port valve and UV-Detector. As a result, the ion-pair reagent was adsorbed to anion

exchange resin and removed.

Recovery, reproducibility and detection limit

The recovery, reproducibility and detection limit of this method are summarized in Table. 1.

The sample concentration was 10 ng/mL and its volume was 100 fj,L. The evaluation was

done based on the peak area of chromatogram. The value of recovery as to VMA was rather

low due to weak retention, but those for others were more than 90%. The values of

reproducibility as to Norepinephrine, Epinephrine and VMA, which are retained weakly in the

pretreatment column, were rather high, but others were within an acceptable level less than

6 /o C.V. Since the retention,times of these compounds fluctuate in the pretreatment process

depending on the pre-saturated ion-pair concentration, the dispersion of reproducibility in the

compounds seemed to be caused by the concentra血n of ion-pair reagent in the internal

surface reversed phase column. The detection limit at signal-to-noise ratio-3 were around 1

1.5 fig/mL. In order to improve sensitivity, micro-LC/MS and Quadruple mass filter

detection can be adopted.

T AB LE. 1

Analysis of the total catecholamine metabolites in plasma

100 (iiL of sample-spiked plasma was analyzed by the present method. Fig. 5 shows the

results. We investigated a variety of compounds in plasma and achieved a clear separation by

HPLC as shown by UV and MS detector. Fig. 6 shows the ion peaks without catecholamine

metabolites. Some of the peaks were identified by both HPLC-UV and MS (Fig. 6a), and the

rest of them were detected only by MS indicating that they had no significant UV absorption

(Fig. 6b).

F IG . 5 , F IG . 6

Conclusion

For analysis of the metabolic process of catecholamines released from cells such as nerve

cells, we established an analytical method of all catecholamine metabolites using

preconcentration column and LC刀MS. We succeeded in separating catecholamine metabolites

and hydrophobic compounds from plasma proteins in a single separation process by using the

internal surface reversed phase column and ion-pair reagent in pretreatment process. In

addition, MS was able to identify some compounds that could not be separated or detected by

HPLC. The recovery were almost 90%, the reproducibility were less than 7% C.V. and the

detection limit at signal-to-noise ratio-3 were around 1 - 1.5 ^ig/rnL. This established method

can be applied to help且nding new markers in Neuroblastoma, by co叩aring the plasma of

patients to that of normal ii血nts. The method can be also used for making a diagnosis of

other diseases or finding their new markers.

Acknowl e dgme山

This work was partly supported by Grant-in-Aids for Scientific Research from the Ministry of

Education, Science, Sports, and Culture of Japan.

Reference

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Fig. 1 The characteristics of the internal surface reversed phase column. Tthis column was selected as a

precolumn for the deproteimzation and a trapping of small molecules.

Fig. 2 The pathway of synthesis and metabolite about catecholamines

( l)Tyrosine-hydroxylase, (2)DOPA-decarboxylase, (S )Dopaminかβ -hydroxylase, (4)PhenylethanolaminかN-

methyltransferase, ( 5 ) catechol -O -methyl -tranceferase, (6) monoamine-oxidase

Fig. 3-a The LC chromatogram. The elution order was Norepinephrine, Epinephrine, Normetanephrine,

Dopamine, Metanephrme, DOPA, VMA, DOPAC, and HVA. Flow rate: 0.2mL/min. The sample

concentration: 10 ng/mL. The sample volume: 20 ^L. Detection: UV280 nm.

Fig. 3 -b MS-TIC四SI-TOE/MS; positive mode)

Fig. 3-c Norepinephrine m/z 152(【M十H -H2O】"*), m/z 170 (【M+H]*)

Fig. 3-d Epinephrine m/z 184 ([M+Hf)

Fig. 3-e Normetanephrine m/z 166 ([M+H -H2O]^ m/z 184 (【M+HJ+)

Fig. 3-f Dopamine m/z 154 ([M+H]つ

Fig. 3-g Metanephrine, DOPA m/z 180 (【M+H -HaOD m伝198 (【M+H]*)

Fig. 3-h DOPAC m/z 169 JM+HT m/z 186 [M+NR,]+ m/z 191 【M+Na]+

Fig. 3-i HVA mた183 【M+HT m/z 200 【M十NELif m/z 205 【M+Naf

Fig. 4-a The LC chromatogram of Sample-spiked FCS. ODS did not hold catecholamine metabolites.

Fig. 4-b The change of proteins concentration. Plasma proteins ran dowm within 3 minutes.

Fig. 4-c The effect of ion-pair reagent. 0. 1血M pentadecafluorooctanoic acid/ 1 % formic acid gave a good

separa也on.

Fig. 4-d A:Injection→3 min. B: After column swifting

Fig. 5-a The LC chromatogram of sample-spiked plasma. We investigated a variety of compounds in plasma

and succeed a clear separation by HPLC-UV detector as to standard compounds.

Fig. 5｣b MS-TIC (ESI-TOP/MS; positive mode)

Fig. 5-c The MS chromatogram. Standerd compounds were detected their peaks respectively.

Fig. 6 The ion peaks without catecholamine metabolites detected by ESI-TOF/MS; positive mode

a Two peaks detected by bo也HPLC-UV and MS.

b Two peaks detected only by MS

Table 1. The evaluation of reproducibility, recovery and detection limit. (n-5)

recovery (% ) m eans S.D . C .V (% ) detection lim its′N =3 (ug/m L )

N orepinephrine 94.1 3 05000 2 14 00 7■0 1

E pinephrine 94 .1 3 `3 000 2 77 00 7.6 1

N orm etanephrin e 97.9 2タ4000 79 00 2■7 1

D opam in e 93.3 3 41000 123 00 3.6 1

M etanephrine 93.3 3 57000 184 00 5●2 1

D O PA 94.0 32 3000 730 0 2.6 1■5

T yrosine 97.4 207 000 7200 3●5 1

Ⅵ止A 87.0 14 5000 1100 0 7■4 ー

D O P A C 9 7.7 350 000 1100 0 3●1 1.5

H V A 98.0 435 000 2600 0 6.0 1

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