Gas chromatography of phenolic and catecholic amines as the trimethylsilyl ethers

Vol. 13, No. 5, 1963

BIOCHEMICAL

GAS

CHROMATOGRAPHY AMINES N.

September

many

(Vanden

other

compounds

by

gas

analysis

of

physiologically these

high

described

a method

for

the

chromatography

on

able

for

This

conmnunication

by

from

reports

by

conversion of et al --•

of

more

to

many

such

as

with of

be

of easily

for

sugars

the by

390

the is

analysis

not

like

the

catecholamines.

chromatographic

suit-

analysis

norepinephrine,

3-methoxytyramine. derivatives,

ethers.

we

could The

Bentley

-et

idea

originated of

(1963).

-*al

by

treatment

in

the

presence

found

be pre-

trimethylsilylation

(HMDS)

a solvent.

gas

it

gas

accomplished

hexamethyldisilazane

for

by

but

catecholamines

(TMS)

method

good

is

recently

amines

acetylated the

(1962)

amines

epinephrine,

and

trimethylsilyl

and

the

This and

Pisano

is

amines

normetanephrine

a convenient

and

ic

for

chromatographic

lacking.

aromatic

phenol

a method

gas 11

1960)

al.,

successfully

nonvolatile

method

complicated

experiments

may with

some

derivatives

sti

et

been

the

Fales

Their

of

for

polar,

of

(Lloyd

have

are

generally

amines,

ine,

(1958).

trimethylsilylation

as

Research, British Columbia,

alkaloids

amines

column. and

volatile

catecholamine

McGeer

methods

separation

amines

unsuccessful

Langer

CATECHOLIC ETHERS

interest

temperatures.

important

suitably

are

describes

metanephr

pared

L.

1960),

al.,

Good

SE-30

analysis

biologically

After that

an

aromatic

dopamine,

P.

biological

amines at

of

and

Sen

important

unstable

the

P.

chromatography.

because

siwle

TRIMETHYLSILYL

et

of

extremely

of

AND

AS THE

Heuvel

analyzed

largely

PHENOLiC

RESEARCH COMMUNICATIONS

26, 1963

Steroids and

OF

Kinsmen Laboratory of Neurological of Medicine, The University of Vancouver, Canada

Faculty

Received

AND BIOPHYSICAL

of

phenols

The the of

pyridine

BIOCHEMICAL

Vol. 13, No. 5, 1963 About

I3

stoppered

my

test

HMDS.

The

ture

and

The

small

of

the

tube

were

mixture then

injected

directly

in

not

columns

and

affect

The

hydroxide,

solved

in

The

injector

200'

C or

but

the

of

different

support

was

Chromosorb

and

C.

excess

reagent

also

the

al.,

was

reacts

treated

analysis

was

amlne,

probably

of

the

TMS derivative

is

not

clear namely,

the

but

the

be

W,

with

l/3

60-80

with

used,

The

inch

internal

mesh,

washed

methanol

SE-30

dual

ic

potassium The

siloxane

under

sol

polymer

vacuum

column

columns

first

effects.

at

temperature produced

dis-

40.

C.

either

separation

of

the

TMS

38 ml/min.

of

the

carrier

et

al.,

i lyl

sharper

by

phenolic

ethers

is

peaks,

derivatives gas

It

was

conditions

gas of

a new

different reacted ic,

phenolic with

alcoholic

all and

391

earl the

derivative

of The

exact

and

catecholic

the

available

amine

alcoholic

that

of

on

derivative.

the

found

stated

chromatography

and

we1 1 establ

A 10 mg portion

the

formation

with

1963).

groups.

under

probably phenol

and

removed

N-trimethylsilyl

HMDS

could

was

Electric

tr imethyls

HMDS

of

the

purification.

adsorptive

of

minutes.

during

equipped

the

tempera-

30

better

amine

the

aliquot

quantitatively

analyzed

revealed

a 1 pl

of

cases.

Bentley

with

formed

ml

room

temperature

reacts

with

additional

reduce

C and

rate

respective

1958;

mixture

all

an

which

at

1.5

column

gave

HMDS

to

290’

A flow

for

then

solvent

higher

in

that

form

reaction

The

hours

length,

and

6% General

was

used

fact

et

centers,

the

HMDS

temperature

was

(Langer

The

in

amines.

to

amine

with

20

detector

solid

glass-

and

for

ionization

acid

a 10 ml

pyridine

chromatograph

in

temperature

The

gas

of

further

6 feet

ether

(nitrogen)

groups

800

without

tubes,

suspended

lower

and

flame

treated

220*

bath

chloride

column

hydrochloric

and was

C water

in

ml ly

steel

concentrated

support

the

model

0.5

analysis,

a differential

diameter. with

the

RESEARCH COMMJNICATIONS

hydrochloride

mechanical

ammonium

onto

stainless

its with

a 95O

of

A Perkin-Elmer

were

shaken

precipitate did

or

treated

was

heated

reaction

amine

AND BIOPHYSICAL

groups.

ished the

phenylethylier,

and

SE-30

the

column.

phenylethylnature amines reactive

id

Vol. 13, No. 5, 1963 All

the

tailing

BIOCHEMICAL TMS derivatives

on this

A typical

times

given

tried,

column

chromatogram

metanephrine tion

SE-30

in Table

and each produced

well

with

a single

in Figure of the

peak.

of epinephrine,

1, and the

different

little

symmetrical

of the TMS derivatives

is shown

TMS derivatives

RESEARCH COMMUNICATIONS

chromatographed

of a mixture

and dopamlne of the

AND BIOPHYSICAL

relative

amines

reten-

studied

are

I.

4r

TMS- Dopamine

Fiqure I. Gas chromatographic separation of t r imethyls i lyl derivatives of amines. Conditions: Column 6 feet x l/8 inch I.D. stainless steel tube, 6% SE-30 on 60-80 Chromosorb W; temperature 220. C, nitrogen flow 38 ml/min.

AS evident epinephrine

could

nor

could

The use of

liquid

colu~l

oil

in Table not the

phases

and sometimes

or to the disappearance

paper

norepinephrine

(Weiss

et al., to the MS

procedure

allowed

In a typical norepinephrine

succinate,

ADS) did led

of peaks.

In phenol

1963),

trlal

from

saturated

for run,

separated

was submitted

1 ug amounts a mixture to paper 392

of

20 M, 5% DC-710

the

separation

lnguishing

was obtained

by

acid

subsequent

on an SE-30

column.

of each of the

amines

1 pg each of epinephrine

chromatography

tails

epinephrlne

1 N hydrochloric amines,

of

of extensive

normetanephrine with

SE-30

and normetenephrine.

Carbowax

In dlst

and nor-

on the

appearance

and chromatography

analysis

other

improve

Success

elut ion of the

derivatives

not

to the

and metanephrine

chromatography

the

of metanephrine

(butanediol

derivatives

of epinephrine

one from

TMS derivatives

L on Anakrom

from

TMS derivatives

be separated

or 5% Apiezon

these

sion

1, the

converThis studied. and

and the separated

BIOCHEMICAL

Vol. 13, No. 5, 1963

AND BIOPHYSICAL

Table Relative

Retention

Times

of

RESEARCH COMMUNICATIONS

1,

Trimethylsilyl

Derivatives

of

6% SE-30

Amines.a

Column

Amine

Dopamine Epinephrine Norep inephr DL-Metanephr DL-Normetanephr 3-Methoxytyramine

ine ine ine

200. c

220.

1.24

1.2

2.04 2.04

1.84 1.84

1.66 1.66

1.56

1.56

(: ::)b

a For

experimental

b Actual

amines

eluted

the

residue

the

conditions

mixture

with

retention

water.

treated

could

be

I ml

previously

to

0.004 the

reading This

ance. No.

609-S-146,

Association

TMS

minutes.

were

a 1:1

Hg of

text.

concentrated

mixture

of

Four

~1

the

amine)

derivative

of

to

pyridine

al iquots

and of

were

dryness

each

and

HMDS

under

react

ion

chromatographed

epinephrine

or

and

norepinephrine

detected. We wish

Acknowledqements. critical

eluants of

(k,“)b

see in

described. to

corresponding

time

The

with

(corresponding

a peak

conditions

c

work

of was N. I.H.

Research

the

to

thank and

manuscript,

supported Grant

by No.

Dr. Mr.

E. L.

G.

Ball

Federal-Provincial

~-5664,

and

McGeer for

for technical

Mental a Canadian

her

Mental

assist-

Health

Grant

Health

Award.

References Bentley, Faies, Langer, Lloyd, Vanden Weiss,

6 iochem. R., Sweeley, C.C., Makita, M., and Wells, W.W., Biophys. ;ies. Comm., .Q., 14 (1963). H.M., and Pisano, J.J., Analyt. Biochem., 1, 337 (1962). S.H., Pantages, P., and Wender, I., Chem. G Ind., 1664 (1958). H.A., Fales, H.M., Highet, P.F., Vanden Heuvel, W.J.A., and Wildman, W.C., J. Am. Chem. Sot., 82, 3791 (1960). Heuvel , W.J.A., Sweeley, C.C., and Horning, E.C., J. Am. Chem. sot., 82, 3481 (1960). B., Rossi, G.V., and Reber, L.A., Nature, m, 281 (1963).

393