Molecular aspects of debrisoquine metabolism studied by gas chromatography mass spectrometry with electron capture negative ion chemical ionisation

Journal of Mass Spectrometry and fan Physics, 48 (1983) 89-92 Elsevier Scientific Publishing Company, Amsterdam - Printed in The NetherIands

89

International

MOLECULAR MASS

ASPECTS

OF DEBRISOQUINE

SPECTROMETRY

S.MURRAY,

G-C-KAHN,

Department Ducane

WITH

ELECTROH

Road,

CAPTURE

A.R.BOOBIS

of Clinical London

METABOLISM

NEGATIVE

BY GAS CHROMATOGRAPHY

ION CHEMICAL

IONISATION

and D.S.DAVIES Royal

Pharmacology,

W12 OHS

STUDIED

Postgraduate

Medical

School,

(UK)

ABSTRACT A gas chromatographic mass spectrometric assay using electron capture negative ion chemical ionisation has been developed for the analysis of 4-hydroxydebrisoquine in human liver microsomal incubations. The assay has been guanoxan and phenformin are competitive inhibitors used to show that sparteine, of human liver debrisoquine 4-hydroxylase activity in vitro.

INTRODUCTION Debrisoquine treatment

of hypertension.

4-hydroxydebrisoquine exhibits

a genetic

graphic vitro

mass

by human

(ref.3,4). enzyme

ment

P-450

limit

has been

used

of

liver

human

oxidation

that

that

We have

this

in man

impact

that the defect

to a deficiency activity,

is reaction

a gas chromato-

(ET)

produced

present,

in

ionisation

in oxidation

or absence

when

in the

hydroxylation

developed

uses electron

this

used

for 4-hydroxydebrisoquine

shown

is due

agent

of

of hepatic

is catalysed

by

(ref.4). assay

ion chemical

in the

which

we have

in vivo and

assay

blocking

of debrisoquine

has shown

(ref.l,2).

microsomes

(ref.3)

metabolite

work

(GC-MS)

this assay,

published

negative

recent

polymorphism

observed

activity

The

and

liver

With

cytochrome

A major

spectrometric

debrisoquine

sympathetic

is a post-ganglionic,

has now

been

ionisation

of detection

to investigate in vitro

polymorphism

modified

to incorporate resulting

{ECNICI),

the effect

on debrisoquine

reportedly

capture

in a substantial

of 4-hydroxydebrisoquine.

of drugs

electron

associated

The modified 4-hydroxylase with

improveassay activity

the debrisoquine

in vivo.

METHODS Sample

Preparation

This

was as described

the final GC-MS

extracts

analysis.

altered

were

for the assay not

Incubations

treated were

the pH to approximately

OOZO-7381/83/000~0000/$03.00

D

using

with

terminated 13.5.

EI ionisation

trifluoroacetic

(ref.4) anhydride

by the addition

A deuterated

analogue

of

except prior

that to

1M NaOH which

of 4-hydroxy-

1983 Elsevier Scientific Publishing Company

90 debrisoquine, After

the internalstandard

a triple

extraction

debrisoquine, debrisoquine converted with

for the assay,

chloroform

the pH of the aqueous and the deuterated

which

phase

analogue

to the corresponding

was

was

then

removed

adjusted

added

most

to 8.5.

in the aqueous

to each

sample.

of the unreacted

phase

4-Hydroxywere

pyrimidinyltetrahydroisoquinolines

then by reaction

hexafluoroacetylacetone.

GC-MS

Conditions

A Finnigan negative

4000

transfer

equipped

with

(100-120

mesh)

gas.

quadrupole

ion chemical

all glass

Under

in the negative tuned

molecular

glass

with

a pulsed

was

used,

gas chromatograph.

column

packed

C with

with

helium

positive

three

minutes.

ionisation

negative

mode with

ions at m/z

(M-) of the derivatives

The mass

The

3% OV-1

methane

via an

latter

of 4-hydroxydebrisoquine

9

as carrier

derivatives was operated

as reagent

371 which

was

on Gas-Chrom

(15 ml/min)

spectrometer

363 and m/z

ion/

interfaced

the pyrimidinyltetrahydroisoquinoline

ion chemical

analogue

9610

at 200 degrees

of about

to monitor ions

deuterated

x 2mm i.d.

conditions,

times

spectrometer

(PPIIdICI) module

to a Finnigan

and operated

these

mass

ionisation

line

a 1.8m

had retention was

with

gas.

were

It

the

and its

respectively.

DISCUSSION 4-Hydroxydebrisoquine problem solved

of extraction by previous

Pnolecule with

(I) is a polar, from an aqueous

workers

(ref.5)

who

hexafluoroacetylacetone

water

soluble

environment

compound.

into an organic

condensed

the amidine

in a two phase

reaction

The consequent solvent

was

sidechain

in the

mixture

(Fig.1).

OH

OH

-

CFa

NH,

I

Fiq.1.

The

The reaction of 4-hydroxydebrisoquine (I) with hexafluoroacetylacetone to give a pyrimidinyltetrahydroisoquinoline (II)

resulting

to partition basis

pyrimidinyltetrahydroisoquinoline readily

into

of an EI ionisation

(II) is sufficiently

the organic

phase.

GC-MS

for 4-hydroxydebrisoquine

assay

This

reaction

has been

non polar used as the

produced

in

9-l vitro

by human

We have

methane

liver

recently

as

microsomes recorded

gas

reagent

(ref.3,4). mass

the ECNICI

spectrum

of compound

II using

(Fig.2).

* ZI

.Z

KY

100

a3

.-c

100 1

200

300

k-7

400

ECNICI

75 I-

^_^

jbj

01 > .-

se

( M- 1

c m

-

I

I 200

I

I 100

L

1 300

I

,I 4Ocl

m I t

Fig.2.

The EI

Besides

possessing this

ion current, the EI

mass

detection

ion

These

(ref.10).

metabolism

liver

as those sparteine of these

microsowes.

of these

drugs

as competitive

II

363 which

as intense

constitutes

as m/z

modifying

in a substantial

344

(the base

the published

improvement

By monitoring

80% of the total peak)

GC-MS

in the limit

negative

ions at m/z

in

assay

of 363, an

to 500 femtograms4-hydroxydebrisoquine

to noise

other

the modified

the effects

of human in vitro

include

Using

a signal

of several

pair of alleles

studied

times

II corresponding

with

of compound

Consequently,

of 4-hydroxydebrisoquine.

of compound

The oxidation quine.

results

spectra

ion at m/z

twenty

is

(ref.6).

ECNICI

can be detected same

mass

a molecular

spectrum

to incorporate

amount

and ECNICI

ratio

drugs

of one

appears

regulating

(ref.6).

to be under

the alicyclic

(ref.7,8),

guanoxan

the control

hydroxylation

(ref.9)

assay

for 4-hydroxydebrisoquine,

three

drugs

on debrisoquine

enzyme

as well

as debrisoquine, of debrisoquine

we

4-hydroxylase

is responsible

inhibitors

of debriso-

and phenfarmin

GC-MS

If the same

of the

then these

have activity

for the oxidative compounds

4-hydroxylation.The

shouldact results

92 we obtained

TABLE

are given

in Table

1.

1

The effects activity

of sparteine,

of human

Drug

guanoxan

and phenformin

on debrisoquine

liver microsomes

Concentration

Debrisoquine

4-hydroxylatioh

Vmax(pmol/mg/min)

(mM)

Phenformin

I I

In

all three

increased remains

rl?sponsible

l?cl 3267 67.33

87 83

0 0.1 0.3

61 63 61

116 434 1554

36 24

1U7 105 113

125 550 1737

220 190

cases,

the Km for debrisoquine

increasing

unchanged.

inhibitors

;Zl 71

2.5

with

(PM)

205 5.0

0 0.75

~

Ki

Km(uM)

Sparteine

Guanoxan

4-hydroxylase

These

of debrisoquine

drug

concentration,

dat a indicate

that

4-hydroxylation

for the oxidative

4-hydroxylation

metabolism

while these

Vmax

three

and suggest of all four

is markedly

for the compounds

reaction are competitive

that the same

enzyme

is

compounds.

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