Permethylation and mass spectrometry of intact ether glucuronides at the low nanomolar level

Vol. 48, No. 5, 1972

BIOCHEMICAL

PERMETHYLATION ETHER

AND

Institute

MASS

GLUCURONIDES R. M. for Baylor

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

AT

Thompson

SPECTROMETRY

THE

LOW

OF INTACT

NANOMOLAR

and D. M.

1

Desiderio

Lipid Research and Department College of Medicine, Houston,

LEVEL

of Biochemistry Texas 77025

Received July 25, 1972 SUMMARY: A group of steroid glucuronides and one drug glucuronide were permethylated with the dimethylsulfinylmethide anion and methyl iodide. All permethylated products were easily recognized as glucuronides from their mass spectra. Overmethylation was observed in those steroid samples which contained isolated or %,a-unsaturated ketones, but this did not hinder the interpretation of the spectra. The technique has further application for the study of drug metabolism. Conjugation anism

(1).

or acid ducts

with

Determination

hydrolysis

e There

are

varying

substrate

sence

of inhibitors.

must

iodide

some

difficulties

about

(GC-MS,

uranic

acid

to some ’

Fellow

2).

intact

acid

by analysis with

hydrolysis

may

and combined et al.,

derivatization

techniques

glucuronides.

Tamura

for

mechenzymatic

of one of the prohydrolysis:

specificity,

and the pre-

be preferable,

the yield

of the glucuronic

technique

upon

enzymatic

in enzyme

acid

but it

of the aglycone

moiety (DMSO-)

the analysis

(1). and

of saccharides

gas chromatography-mass have

in polysaccharides

Copyright @ 1972 by Academic Press, Inc. All rights of reproduction in any form reserved.

mainly

dimethylsulfinylmethylsodium

Bjblrndal,

of the Intra-Science

detoxification

relied

a way as to maximize

is a useful (GC)

residues

Various

differences

degradation with

(CH31)

metry

theoretical

Therefore,

gas chromatography

has

followed

Permethylation methyl

is a well-studied

of the conjugates

out in such

concern

acid

of glucuronides

affinities,

be carried

without

glucuronic

reported

by

spectro-

the permethylation

of

(2). and GC have and Imanau

Research

1303

Foundation,

previously

were

able

1971-

been

applied

to chromato-

1975.

Vol. 48, No. 5, 1972

graph

both

BIOCHEMICAL

methyl

The

use of acetyl

Both

DMSO-/CH31

ation.

Less

product GC.

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

and trimethylsilyl derivatives

(TMS)

was

reaction

occurred

Trimethylsilylation that

A GC-MS

analysis

has been (8) have be handled

(6). that

were

separated two-step

esterified

recently

permethyl-

procedures

with

Billets,

et al.,

of certain

intact

One

side

and identified

by

which

diazomethane

of 17-ketosteroid

TMS-derivatives

to report

here

by permethylation

introduction

that

intact

ether

and mass

(4, 5).

glucuronides

(7) and Horning drug

et al.,

glucuronides

can

glucuronides

spectrometry

can be easily

through

the direct

system. MATERIALS

The

for

was the solvent.

was

be initially

More

employed

(4,5).

by GC and CC-MS.

We wish identified

compound,

acid

glucuronides

were DMF

of the TMS-derivatives

reported shown

when

and acetylation

the uranic

of g-glucuronides.

to simple

systems

( < 100/o), the 4-dehydro

required

limited

and DMF-/CH31

side

derivatives

following

Laboratories,

steroid

Inc.,

New

methyl

cortisone-2la-glucuronide, 17e-glucuronide,

METHODS

glucuronides

Denville,

glucuronide-tri-g-acetyl

AND

were

Jersey,

ester,

obtained 07834:

from

Research

A16-androstene-3a-

Plus -

androsterone-32-glucuronide,

pregnanediol-3a-glucuronide,

testosteroneThese

and tetrahydrocortisol-3a-glucuronide.

were

used

as received. The ramidol,

glucuronide see Fig.

of a-[(Z-pyridylamino)-methyl] 4) was

a gift

the Department

of Pharmacology,

A 10 pg sample

of each

2

The g-acetyl

groups

was

are

from

Dr. Baylor

permethylated

changed

benzyl

N.

Gerber

College

1304

(pheny-

and Dr.

R. Seibert

of Medicine,

Houston.

by the technique

to Q-methyl

alcohol

during

of Leclercq

derivatization.

of

and

BIOCHEMICAL

Vol. 48, No. 5, 1972

Desiderio

for

pretation

of the

(ca.

2-5

plete the

peptides

of original

and low

with

either

16 or

eratures

often

be

70

ranged

eV

from

yielded (M-31)+,

and

highest

mass

ment

types

of mass

those

containing

ions.

Figures

illustrate

is

the

not

1-2

pumped

obtained

give

a com-

readily

from

an

LKB-9000

ionizing

potential

inter-

introduction.

on The

in

micrograms

to

after

probe.

AND

3.5

mass

potential

kV.

Source

temp-

DISCUSSION

glucuronides: spectra:

each

plus

glucuronide

and

3,

number

of

added

the

and of these

it

Mt,

spectra

testosterone-

compounds

those

containing as

the

of permethylated 17e-glucuronides

Indicated

contains

these

fragments

mass

types.

methyls

general,

containing

steroid 1,2,

In

those

pregnanediol-3a-,

respectively,

of

hours

accelerating

steroid

androsterone-3a-,

several were

to help

270-290°.

Permethylated three

used

material

5 pg were

introduction the

were

sufficient

than

RESULTS 1)

was

spectra

direct and

CD31

equivalent3

detected

mass the

and

permethylated

greater

resolution

spectrometer was

could

CH31

sample

Samples

spectrum.

All

The

spectra.

nmoles)

source

Both

(3).

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

for

as

each

major

determined

by

frag-

perdeu-

te riomethylation. The

most

compounds ions

is

common from

3 This washing

is always to

striking the the

all

fragmentation

assumes of the

feature

pair

of

most

spectra

which

ions

at --m/e

intense

ion

is

common

101 in

each

of permethylated of the

complete products.

--m/e

the

acid

and

no

1305

spectra

201.

spectrum.

One

of these of these

two

they

are

Because

glucuronides,

glucuronic

reaction

and

to

these

moiety

and

losses

during

may

ions be

extraction

must

represented

and

arise by

Vol. 48, No. 5, 1972

the following

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

structures:

CH30-CH=CH-CH=&CH3 (m/e -Upon

101)

(m/e --

perdeuteriomethylation,

respectively,

and this

as shown.

Therefore,

identification

spectra

are

methylated

ions

steroid

nide

(MW=566,

201 peaks

can be used

glucuronides

providing

there

There

are

no ions

analogous

ions methyl

2),

in the Mt

Most

Mf

ions

due to the steroid

Fig.

added.

bond

with

1, --m/e

In the case mass

region

Figure

the charge 317 in Fig.

present.

217 and --m/e

317 ions

in the

When

in the mass

is at --m/e

3 also

of per-

indicate

testosterone

535,

not show 520,

mono, probably

(-OCH3).

was di,

andros-

an Mt

but does

as many

itself

Mt-31

of permethylated

does

at --m/e

components:

spectra

of pregnanediol-3a-glucuro-

ion

and 576 which

of the overmethylation

moiety.

the

peaks

also

of the spectrum

1).

of five

are

are

groups

for

glucuronide

present

17e-glucuronide

562,

is an a, S-unsaturated While

side

548,

was a mixture

methylated. there

are

not always

the highest

(Fig.

534,

groups

product

are

testosterone-

at --m/e

to the --m/e

methyl

(7).

(M’)

terone-3a-glucuronide permethylated

or of the entire

and three

210

101 and --m/e

glucuronides.

Fig.

107 and --m/e

the --m/e

derivatives

Molecular

to --m/e of two

probably

of TMS

shift

the presence

of the aglycone

ions

ions

indicates

of permethylated

representative These

these

201)

ion for

show

as four

MS.

extra

permethylated, tri,

occurs

tetra,

the

and penta-

on ring

A,

where

ketone. not always These

present, peaks

remaining 2, and --m/e

every arise

from

on the aglycone: 285,

1306

299,

spectrum cleavage --m/e

contains

peaks

of the glyco273 and

313 and 327 in Fig.

287 in 3.

It is

k _1 2

-

25.

IOOz 9 75 5; s 50 2

FIGURE

I

2

.I. 100

16eV

lOl(2)

MASS

I

I

70eV MASS ( ) INDICATE

.

SPECTRUM

150

, i

itr\

Og @-3

\a?? \ o 0”s

(0) 257 L. 250

(0) 303 I 300

m/e PREGNANEDIOL

(0) 285 (II 317

0

MW=522

I-CYOH

(0)

170

EV.120

1 363 I 1 350

(,)

-3d-GLUCURONIDE .

(2) L

I 1 1

E2 II (2) 377

(16

I

(2) 391

-m/e- K

OH

ADDED

v50

DEG1

DEGI

( ) INDICATE

I Ltoo

EV.lYO

ANDROSTERONE-30(-GLUCURONIDE

261

PREGNRNEDIOL-3a-GLUCURONIDE

m/e SPECTRUM OF PERMETHYLATED ADDED METHYLS

cyo

coc-?3

273

RNDROSTERONE-3d-GLUCURDNIDE

OF PERMETHYLATED

(3) 201 , , 200

PERMETHYLRTED

FIGURE

PERMETHYLQTED

METHYLS

(4) 505 500

M-OCH, (41 535 c

I

18

36.6

w .x

w

Y

5

c

2

z e t

z

25.

50

75

100.

7OeV

FIGURE

50

FIGURE3:

4

(0) 70

70eV

MASS

100

MASS

(2) 150

200

RIENYRAMIDOL

250

GLUCUAONIDE

300

I

I I I (4)

(70

178 -GLUCURONIDE

D PHENYRAMIDOL

OF PERMETHYLATED

169

(2)

PERMETHYLRTE

m/e TESTOSTERONE-

TESTOSTERONE-17b-GLUCURONIDE

OF PERMETHYLATED

SPECTRUM

121 (II

SPECTRUM

PERMETHYLRTED

I-

350

( 1 INDICATES

m/r 201 =g&

CH,ctl

UOO

169

OEGI

( 1 INDICATE

EV.138

ADDED

W).M-CyOH

ADDED

METHYLS

950

(5l.M' 460

METHYLS

1

-19

; w S

- 37 .a

BIOCHEMICAL

Vol. 48, No. 5, 1972

obvious the

that

androsterone-3a-glucuronide

second

under

cleavage

these

Fig.

but

not

at --m/e

- --m/e

--m/e

cleavage

ions

peaks

are

of

on

the

little

in

386,

itself

Fig.

spectra

diagnostic

and

400

in

of

overmethylated

acid

base

+ 30,

for

the

363,

Fig.

377,

and

391

3 - probably

moiety, base

+ 44,

of permethylated

value

because is

1; m/e --

glucuronic to the

mass

overmethylated

D.

358

373,

analogous, in

ring

347,

358,

also

Androsterone

still-attached

necessarily

t 70

287.

333,

343,

of the

base

is

probably

peaks

2; and

from

ion

conditions,

Certain in

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

and

are

base

t

nucleosides

identification

of

arise similar, 58,

and

(9).

These

permethylated

glucuronides. Unfortunately, mentation

of the

methylated identify

any

individual

indicated

are

not

due

to

so

ify

that

lated of

other

(except identify

possibly it

of the

conclusion,

if

permethylated

one

as We

are

be

frag-

of a per-

used

the

to

to

positively

masses

of

some

Figure

very

All

the

the

major

101

ion

at --m/e

121

clearly

identified

spectrum and

was

and

used

--m/e

as

mass

cleavages

--m/e

intense This

4 shows

201 which

proof

ions is the

of

(10). is

familiar

aglycone

compound

yet

due

a glucuronide.

of phenyramidol

metabolite

spectrum

from

case,

aglycone.

glucuronide

a mass

glucuronide.

In this

because

ions

glucuronide:

spectrum.

of the

the

as

significant

not

phenyramidol

significant

of this

sample.

probably

phenyramidol

on the

as

structure

the

to

fragmentation

compound

In

steroid

but-only

contain

Therefore, can

of permethylated

are

not

nucleus.

Permethylated

spectrum

do

glucuronide

ions),

2)

spectra

steroid

steroid

cleavage

of

the

a glucuronide employing

with

portion

the

mass

spectral

of a glucuronide, from

this

the technique

glucuronides.

1309

mass

fragmentation he

spectrum further

can

readily

of a permethyfor

the

identification

ident-

BIOCHEMICAL

Vol. 48, No. 5, 1972

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

ACKNOWLEDGMENTS The authors are grateful for financial of General Medical Sciences (GM- 13901, were supported by NIH RR 254 and 259.

support from the National Institutes GM-02055). Computer facilities

REFERENCES 1.

Dutton, Academic

Geoffrey Press,

J., ed., Glucuronic Acid: Free and Combined, New York and London, 1966, 629 pp.

2.

Bjgrndal, Svensson,

Hgkan, Sigfrid,

3.

Leclercq,

P. A.

4.

Tamura, (1964).

Zenzo

5.

Imanari, (1967).

Toshio

6.

Hashimoto, and Masuda,

7.

Billets, Stephen, Tietman, Paul S., and Fenselau, Catherine, Abstracts Twentieth Annual Conference on Mass Spectrometry Allied Topics, Dallas, Texas, June 1972.

Hellerqvist, Angew.

and Desiderio, and Imanari,

and Tamura,

D.

M.,

Anal.

Lindberg, Bengt, and Edit., 2, 610 (1970). Lett.,

4,

Horning, Twentieth Dallas,

9.

von Minden, David Annual Conference Texas, June 1972.

Chem.

Pharm.

Bull.

12,

1386

Zenzo,

Chem.

Pharm.

Bull.

15,

1677,

M. G., Harvey, D. J., and Horning, E. Annual Conference on Mass Spectrometry Texas, June 1972. L. and McCloskey, on Mass Spectrometry

Gerber, N., Thompson, Abstracts, Fifth Internat. July, 1972.

305 (1971).

Toshio,

Keizi, Inoue, Takehisa, Masaki, Kiyotaka, Yoshiro, Bunseki Kaguka 2, 1607 (1970).

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10.

Carl Gustaf, Chem. internat.

Fukui,

Iwao,

and

C., Abstracts and Allied Topics,

James A., Abstracts and Allied Topics,

Twentieth Dallas,

R. M., Seibert, R., and Desiderio, D. M., Congress on Pharmacol., San Francisco,

1310