Identification

of

sulfation

sites

of

metabolites

and

prediction

of

the

compounds

biological

effects

Characterizing

the

biological

effects

of

metabolic

transformations

-LRB-

or

biotransformation

-RRB-

is

one

of

the

key

steps

in

developing

safe

and

effective

pharmaceuticals

Sulfate

conjugation

one

of

the

major

phase

II

biotransformations

is

the

focus

of

this

study

While

this

biotransformation

typically

facilitates

excretion

of

metabolites

by

making

the

compounds

more

water

soluble

sulfation

may

also

lead

to

bioactivation

producing

carcinogenic

products

The

end

result

excretion

or

bioactivation

depends

on

the

structural

features

of

the

sulfation

sites

so

obtaining

the

structure

of

the

sulfated

metabolites

is

critically

important

We

describe

herein

a

very

simple

high-throughput

procedure

for

using

mass

spectrometry

to

identify

the

structure

and

thus

the

biological

fate

of

sulfated

metabolites

We

have

chemically

synthesized

and

analyzed

libraries

of

compounds

representing

all

the

biologically

relevant

types

of

sulfation

products

and

using

the

mass

spectral

data

the

structural

features

present

in

these

analytes

can

be

reliably

determined

with

a

success

rate

This

work

represents

the

first

example

of

a

high-throughput

analysis

that

can

identify

the

structure

of

sulfated

metabolites

and

predict

their

biological

effects

Introduction

The

study

of

drug

metabolism

is

one

of

the

critical

steps

in

drug

development

-LSB-

-RSB-

At

physiological

conditions

drugs

undergo

a

variety

of

biotransformations

which

produce

metabolites

with

different

chemical

structures

The

resulting

metabolites

differ

in

both

pharmacological

and

toxicological

properties

compared

to

their

parent

drugs

-LSB-

-RSB-

Therefore

to

identify

and

characterize

the

structures

and

properties

of

drug

metabolites

formed

in

vivo

an

extensive

investigation

of

the

structures

generated

by

biotransformation

is

required

Sulfate

conjugation

-LRB-

sulfation

-RRB-

is

one

of

the

most

important

phase

II

reactions

that

occurs

during

the

biotransformation

of

a

variety

of

structurally

diverse

endogenous

compounds

xenobiotics

and

drugs

-LSB-

-RSB-

During

this

process

a

sulfonate

moiety

-LRB-

-RRB-

is

transferred

from

the

donor

phosphosulfate

-LRB-

PAPS

-RRB-

to

the

substrates

through

the

catalysis

of

sulfotransferase

enzymes

resulting

in

sulfated

products

-LSB-

-RSB-

Sulfation

is

generally

considered

as

a

detoxification

pathway

because

the

sulfated

products

are

more

water-soluble

this

facilitates

their

elimination

from

the

body

-LSB-

-RSB-

However

sulfation

can

also

lead

to

the

bioactivation

of

certain

type

of

compounds

which

include

benzylic

allylic

alcohols

and

aromatic

hydroxylamines

Sulfated

products

of

these

compounds

can

undergo

loss

of

resulting

in

reactive

electrophilic

carbocation

or

nitrenium

ion

intermediates

which

covalently

bind

to

cellular

macromolecules

leading

to

mutagenicity

and

carcinogenicity

-LSB-

-RSB-

The

production

of

the

electrophilic

intermediates

relies

on

the

structural

feature

of

sulfation

sites

as

shown

in

Fig.

Under

physiological

conditions

the

sulfate

anions

in

substrates

a

c

in

Fig.

act

as

good

leaving

groups

to

form

resonance-stabilized

intermediates

-LSB-

-RSB-

For

the

other

types

of

sulfated

products

in

Fig.

this

process

is

not

favorable

due

to

instability

of

cation

products

Fig.

of

forming

bioactive

carbocation

or

nitrenium

ion

intermediates

from

sulfated

products

Substrates

a

b

and

c

undergo

the

bioactivation

process

while

d

g

do

not

a

sulfated

benzylic

alcohol

b

sulfated

allylic

alcohol

c

sulfated

aromatic

hydroxylamine

d

-RRB-

sulfated

aliphatic

alcohol

e

sulfated

phenol

f

sulfated

aliphatic

amine

g

sulfated

aromatic

amine

The

biological

sulfation

of

tamoxifen

is

one

example

that

demonstrates

how

the

sulfation

site

dictates

the

biological

fate

of

the

molecule

Tamoxifen

is

a

pharmaceutical

that

is

widely

used

in

the

treatment

and

prevention

of

breast

cancer

It

is

hydroxylated

during

phase

I

metabolism

to

generate

two

products

-LRB-

-RRB-

and

alpha-hydroxytamoxifen

-LRB-

α-OH-TAM

-RRB-

The

sulfation

of

and

that

of

α-OH-TAM

have

completely

different

biological

effects

The

sulfation

of

which

occurs

at

a

phenol

site

-LRB-

as

in

Fig.

-RRB-

leads

to

detoxification

whereas

the

sulfation

of

α-OH-TAM

which

involves

sulfation

of

a

benzylic

alcohol

-LRB-

as

in

Fig.

-RRB-

leads

to

bioactivation

-LSB-

-RSB-

This

example

-LRB-

along

with

many

others

-RRB-

demonstrates

the

fact

that

the

biological

effects

of

sulfation

can

be

determined

by

characterizing

the

sulfation

site

of

the

substrate

It

is

widely

known

that

aromatic

or

aliphatic

hydroxyl

and

amine

groups

are

the

major

sites

in

molecules

that

can

be

sulfated

during

metabolism

-LSB-

-RSB-

However

identifying

the

sulfation

sites

can

be

difficult

because

many

potential

sulfation

sites

can

be

produced

or

modified

during

phase-I

biotransformations

-LSB-

-RSB-

For

example

hydroxyl

groups

can

be

added

to

a

benzene

ring

or

carbon

chain

through

aromatic

or

aliphatic

hydroxylation

generating

new

potential

sulfation

sites

as

in

the

tamoxifen

example

An

amine

group

can

be

changed

to

a

hydroxylamine

group

through

N-oxidation

-LSB-

-RSB-

which

modifies

the

structural

feature

of

the

potential

sulfation

site

Since

these

new

sites

are

formed

in

vivo

characterizing

the

biological

effect

of

their

sulfation

can

be

problematic

It

is

thus

essential

to

develop

a

method

that

can

characterize

the

sulfation

sites

of

unknown

sulfated

metabolites

With

this

information

the

biological

effect

of

sulfation

can

be

estimated

and

the

properties

of

sulfated

metabolites

can

be

characterized

Analytical

techniques

such

as

NMR

spectroscopy

and

mass

spectrometry

have

been

utilized

to

identify

sulfated

metabolites

-LSB-

-RSB-

NMR

is

a

powerful

technique

that

can

not

only

detect

sulfated

metabolites

-LSB-

-RSB-

but

also

help

to

determine

the

position

of

sulfation

in

the

substrate

molecules

-LSB-

-RSB-

However

due

to

the

relatively

high

sample

requirements

the

complexity

of

data

analysis

and

the

need

to

characterize

purified

compounds

the

application

of

NMR

in

identification

and

characterization

of

drug

metabolites

is

limited

in

high-throughput

metabolite

profiling

Compared

to

NMR

mass

spectrometry

is

more

widely

used

in

drug

metabolite

identification

due

to

its

high

selectivity

and

sensitivity

low

detection

limit

and

ability

to

analyze

mixtures

-LSB-

-RSB-

It

is

known

that

mass

spectrometry

can

identify

sulfation

by

detecting

the

Da

mass

increase

in

MS

mode

or

the

characteristic

ions

in

MS/MS

mode

-LSB-

-RSB-

However

limited

information

about

structural

features

of

sulfation

sites

currently

can

be

obtained

from

MS/MS

analysis

with

the

well-known

exception

that

the

product

ion

m/z

-LRB-

-RRB-

can

be

used

to

differentiate

alicyclic

sulfates

from

aromatic

sulfates

-LSB-

-RSB-

In

order

to

facilitate

the

identification

of

sulfation

sites

in

pharmaceuticals

we

developed

a

mass

spectrometry-based

protocol

that

differentiates

between

the

biologically

relevant

sulfation

sites

After

completing

the

studies

on

several

classes

of

sulfated

products

a

set

of

rules

was

developed

to

predict

the

sulfation

sites

With

these

prediction

rules

structural

feature

of

sulfation

sites

can

be

determined

by

detecting

MS/MS

fragmentation

pathways

of

their

corresponding

sulfated

products

With

the

structural

information

of

sulfation

sites

the

two

key

different

biological

effects

of

sulfation

detoxification

and

bioactivation

can

be

differentiated

based

on

literature

precedence

that

links

the

type

of

sulfation

to

its

biological

fate

Based

on

that

information

relevant

properties

of

sulfated

products

can

be

estimated

Experimental

Reagents

Sulfates

and

sulfamates

which

include

sulfate

dipotassium

salt

L-ascorbic

acid

dipotassium

salt

indoxyl

sulfate

potassium

salt

β-estradiol

sodium

salt

potassium

sulfate

β-Estradiol

sodium

salt

sulfate

potassium

salt

sulfate

potassium

salt

hydrogen

sulfate

poly

-LRB-

vinyl

sulfate

-RRB-

potassium

salt

D-glucose

potassium

salt

chondroitin

disaccharide

sodium

salt

N-acetylglucosamine

sodium

salt

N-cyclohexylsulfamic

acid

acid

monopotassium

salt

D-glucosamine

sodium

salt

and

butyl-sulfamic

acid

were

purchased

from

Sigma-Aldrich

-LRB-

St.

Louis

MO

-RRB-

One

sulfamate

sulfamic

acid

was

purchased

from

Scientific

Exchange

Inc.

-LRB-

Center

Ossipee

NH

-RRB-

-LRB-

R

-RRB-

-LRB-

-RRB-

α-phenethylsulfamic

acid

was

purchased

from

Norse

Laboratories

-LRB-

Newbury

Park

CA

-RRB-

The

benzylic

alcohols

allylic

alcohols

hydroxylamines

which

include

-LRB-

R

-RRB-

-LRB-

-RRB-

benzyl

alcohol

-LRB-

R

-RRB-

alcohol

alcohol

alcohol

alcohol

crotyl

alcohol

furfuryl

alcohol

N-methylhydroxylamine

hydrochloride

N-isopropylhydroxylamine

hydrochloride

N-cyclohexylhydroxylamine

hydrochloride

N

N-diethylhydroxylamine

and

N-benzoyl-N-phenylhydroxylamine

were

purchased

from

Sigma-Aldrich

-LRB-

St.

Louis

MO

-RRB-

These

benzylic

alcohols

allylic

alcohols

and

hydroxylamines

were

utilized

to

synthesize

their

corresponding

sulfated

products

The

other

reagents

used

in

the

sulfation

of

alcohols

and

hydroxylamines

were

also

purchased

from

Sigma-Aldrich

-LRB-

St.

Louis

MO

-RRB-

Sulfation

of

benzylic

allylic

alcohols

and

tertiary

hydroxylamines

Sulfated

products

were

prepared

based

on

slight

modifications

to

a

previous

protocol

-LSB-

-RSB-

by

dissolving

equivalent

-LRB-

mmol

-RRB-

of

the

substrate

in

ml

dimethylformamide

-LRB-

DMF

-RRB-

and

adding

sulfur

trioxide-dimethylformamide

complex

-LRB-

mmol

-RRB-

and

pyridine

-LRB-

mmol

-RRB-

The

reaction

mixture

was

stirred

at

°C

for

h.

-LRB-

ml

-RRB-

was

added

to

the

product

solution

and

the

product

was

extracted

with

mL

ethyl

acetate

-LRB-

EtOAc

-RRB-

Evaporation

of

the

solvent

afforded

the

corresponding

sulfated

product

Sulfation

of

secondary

hydroxylamines

To

a

dry

THF

-LRB-

ml

-RRB-

solution

of

NaH

-LRB-

mmol

-RRB-

was

added

secondary

hydroxylamines

-LRB-

mmol

-RRB-

at

°C

Then

the

solution

was

stirred

at

room

temperature

for

h

followed

by

reaction

with

-LRB-

mmol

-RRB-

overnight

The

corresponding

sulfated

products

were

obtained

by

evaporation

of

THF

Sample

pretreatment

Sulfated

compounds

that

were

purchased

were

dissolved

to

a

final

concentration

of

M

with

MeOH

and

directly

injected

into

the

mass

spectrometer

Sulfated

benzylic

and

allylic

alcohols

and

tertiary

hydroxylamines

were

prepared

by

diluting

μl

of

the

liquid

product

by

fold

with

and

then

injected

to

the

mass

spectrometer

Sulfated

secondary

hydroxylamines

were

prepared

by

dissolving

mg

solid

product

in

ml

MeOH

followed

by

injection

into

the

mass

spectrometer

Mass

spectrometry

The

mass

spectra

were

recorded

using

a

Quattro

Ultima

-LRB-

Waters

Corp.

Milford

MA

-RRB-

triple

quadrupole

mass

spectrometer

equipped

with

an

electrospray

ionization

source

Data

was

acquired

in

the

negative

ion

mode

using

a

capillary

voltage

of

kV

and

a

cone

voltage

was

V

The

source

temperature

and

desolvation

gas

temperature

were

and

°C

respectively

Argon

is

used

as

the

collision

gas

and

the

pressure

in

collision

cell

is

E

-LRB-

-RRB-

mbar

The

collision

energy

applied

in

MS/MS

for

all

the

samples

was

eV

Results

and

discussion

The

structures

of

the

sulfated

products

are

in

Fig.

These

compounds

are

representative

of

different

types

of

sulfated

products

that

are

produced

by

sulfation

at

different

sites

Mass

spectrometric

studies

were

conducted

in

the

negative

ion

mode

since

sulfated

products

are

deprotonated

at

physiological

pH

-LSB-

-RSB-

The

common

product

ions

from

the

MS/MS

experiments

are

summarized

in

Table

As

the

table

indicates

common

product

ions

include

m/z

-LSB-

-RSB-

m/z

m/z

and

m/z

The

presence

of

m/z

-LSB-

-RSB-

m/z

or

m/z

ions

are

quite

dependent

on

the

site

of

sulfation

The

presence

of

the

negative

ion

with

m/z

however

is

common

in

the

fragmentation

of

almost

every

type

of

sulfated

products

so

it

is

less

helpful

in

identification

of

sulfation

sites

Thus

this

ion

m/z

is

used

to

identify

sulfation

sites

when

no

other

characteristic

ions

can

be

detected

Based

on

the

MS/MS

data

each

type

of

sulfated

product

possesses

a

characteristic

fragmentation

pathway

which

is

described

below

An

example

of

MS/MS

data

for

each

type

of

sulfated

product

is

provided

in

Fig.

with

the

characteristic

ion

labeled

by

an

asterisk

Fig.

of

selected

sulfated

compounds

a

sulfated

aromatic

alcohols

or

enols

b

sulfated

aliphatic

alcohols

with

β

hydrogens

on

c

sulfated

benzylic

or

allylic

alcohols

d

sulfated

hydroxylamines

e

sulfated

aminesFig

-LRB-

-RRB-

ESI-MS/MS

data

for

compounds

in

this

study

a

sulfated

aromatic

alcohol

from

group

a

b

sulfated

aliphatic

alcohol

with

β

hydrogen

on

from

group

b

c

sulfated

benzylic

alcohol

from

group

c

d

sulfated

aliphatic

hydroxylamine

from

group

d

e

sulfated

aromatic

hydroxylamine

from

group

d

f

sulfated

aliphatic

amine

from

group

e

g

sulfated

aromatic

amine

from

group

eTable

fragmentation

of

selected

sulfated

productsGroupNo.Chemical

nameAbundance

of

characteristic

ions

in

MS/MS

-LRB-

-RRB-

m/z

-LSB-

-RSB-

m/z

acid

hydrogen

sulfate

-LRB-

vinyl

sulfate

-RRB-

disaccharide

α-methyl

sulfate

sulfate

-LRB-

R

-RRB-

sulfate

sulfate

sulfate

sulfate

crotyl

sulfate

sulfate

acid

hydroxylamine-O-sulfonic

acid

68100a5.124N-cyclohexyl-hydroxylamine-O-Sulfonic

acid

27100a1125N,N-diethyl-hydroxylamine-O-sulfonic

acid

acid

acid

acid

acid

acid

-LRB-

R

-RRB-

-LRB-

-RRB-

α-phenethylsulfamic

acid

A

threshold

of

is

used

for

the

relative

abundance

of

characteristic

ions

The

long

dash

-LRB-

-RRB-

means

that

the

characteristic

ion

can

not

be

detected

or

the

relative

abundance

is

below

aRepresents

the

specific

characteristic

ion

of

each

group

that

can

help

to

identify

the

sulfation

site

Group

a

sulfated

aromatic

alcohols

or

enols

Compounds

are

sulfated

aromatic

alcohols

or

enols

For

these

compounds

the

sulfate

group

is

attached

to

an

carbon

-LRB-

benzene

ring

or

double

bond

-RRB-

These

sulfated

products

share

the

same

fragmentation

pathway

by

undergoing

the

neutral

loss

of

Da

and

forming

a

characteristic

ion

at

m/z

-LSB-

-RSB-

as

seen

in

Fig.

After

undergoing

the

neutral

loss

of

phenoxide

or

enolic

anions

are

formed

and

the

negative

charge

is

resonance

stabilized

as

shown

in

Scheme

-LSB-

-RSB-

Scheme

aromatic

alcohols

or

enols

-LRB-

group

a

-RRB-

dissociate

to

produce

a

characteristic

ion

with

neutral

loss

of

Da

Group

b

sulfated

aliphatic

alcohols

with

β

hydrogens

on

The

second

group

of

sulfated

products

originates

from

the

sulfation

of

aliphatic

alcohols

compounds

For

these

species

the

sulfate

group

is

attached

to

an

carbon

All

sulfated

products

of

this

type

can

produce

the

characteristic

ion

m/z

in

MS/MS

and

one

example

is

shown

in

Fig.

The

ion

m/z

is

generated

when

the

proton

from

the

β

carbon

is

transferred

to

the

sulfate

moiety

and

the

C-O

bond

is

broken

to

form

a

bisulfate

anion

-LSB-

-RSB-

See

Scheme

Scheme

aliphatic

alcohols

-LRB-

group

b

-RRB-

produce

the

characteristic

ion

m/z

This

rearrangement

occurs

via

an

energetically

accessible

six-membered

ring

transition

state

and

a

stable

neutral

product

is

formed

As

a

result

this

fragmentation

is

predominant

in

the

MS/MS

data

of

this

group

of

sulfated

products

The

mechanism

shows

that

the

availability

of

a

β

hydrogen

on

an

carbon

is

required

for

this

fragmentation

Unlike

compounds

in

group

a

neutral

loss

of

Da

is

not

observed

from

this

group

of

compounds

This

is

likely

due

to

the

fact

that

the

product

ion

that

would

be

generated

from

loss

of

Da

-LRB-

-RRB-

is

not

resonance

stabilized

so

the

loss

is

not

favorable

Compound

is

a

special

case

because

it

gives

a

characteristic

ion

with

m/z

in

addition

to

m/z

The

relevant

mechanism

for

this

loss

is

addressed

in

the

discussion

of

compounds

in

group

d.

Group

c

sulfated

benzylic

or

allylic

alcohols

In

the

third

group

of

sulfated

compounds

-LRB-

-RRB-

each

contains

a

sulfate

group

attached

to

benzylic

or

allylic

carbon

All

the

compounds

in

this

group

produce

a

characteristic

ion

with

m/z

An

example

of

an

MS/MS

data

for

this

type

is

shown

in

Fig.

The

characteristic

ion

is

produced

by

homolytic

cleavage

producing

benzylic

or

allylic

radicals

The

radical

is

resonance

stabilized

which

is

demonstrated

in

Scheme

-LSB-

-RSB-

Scheme

benzylic

and

allylic

alcohols

-LRB-

group

c

-RRB-

produce

the

characteristic

ion

m/z

In

addition

to

m/z

the

negative

ion

m/z

can

also

be

obtained

for

some

of

the

compounds

in

this

category

For

example

compounds

and

both

have

an

abundant

peak

at

m/z

due

to

the

availability

of

a

hydrogen

on

the

carbon

in

close

proximity

to

the

sulfate

group

Even

though

compound

is

a

benzylic

sulfate

a

hydrogen

attached

to

the

β

carbon

is

present

Therefore

just

like

compounds

in

group

b

it

undergoes

a

rearrangement

to

form

the

ion

m/z

Compound

possesses

a

methyl

group

in

the

ortho

position

and

the

availability

of

hydrogen

attached

to

the

carbon

in

methyl

group

enables

a

similar

rearrangement

-LRB-

eight-membered

ring

-RRB-

to

form

the

product

ion

m/z

as

illustrated

in

Scheme

Even

though

β

hydrogens

are

available

in

other

benzylic

and

allylic

sulfates

in

this

group

the

fragmentation

to

produce

m/z

is

not

as

favorable

since

the

hydrogens

are

on

the

hybridized

carbons

The

neutral

loss

of

Da

is

not

favorable

either

because

the

product

ion

that

would

be

generated

from

such

a

loss

is

not

resonance

stabilized

Scheme

mechanism

producing

m/z

from

compound

Group

d

sulfated

hydroxylamines

The

fourth

group

of

sulfated

products

originates

from

sulfation

of

hydroxylamines

and

as

a

result

the

sulfate

group

is

directly

attached

to

the

amine

See

compounds

All

the

sulfated

products

of

this

group

can

produce

the

characteristic

ion

m/z

and

examples

of

MS/MS

data

are

shown

in

Fig.

A

characteristic

ion

with

m/z

can

be

obtained

for

sulfated

products

originating

from

both

aliphatic

-LRB-

Fig.

-RRB-

and

aromatic

-LRB-

Fig.

-RRB-

hydroxylamines

For

these

sulfated

compounds

homolytic

cleavage

is

favorable

This

is

due

to

the

small

electronegativity

difference

-LRB-

about

-RRB-

between

N

and

O

compared

to

electronegativity

difference

of

around

for

C

N

and

O-S

-LSB-

-RSB-

The

small

electronegativity

difference

causes

the

electrons

to

be

split

equally

-LRB-

homolytic

cleavage

-RRB-

between

the

oxygen

and

nitrogen

when

the

bond

breaks

instead

of

both

electrons

moving

onto

the

oxygen

or

nitrogen

which

would

be

the

case

during

heterolytic

cleavage

In

Scheme

the

mechanism

of

this

homolytic

cleavage

is

depicted

An

amine

radical

is

formed

as

the

product

Scheme

hydroxylamines

-LRB-

group

d

-RRB-

produce

the

characteristic

ion

m/z

This

mechanism

can

also

be

used

to

explain

the

presence

of

the

ion

with

m/z

in

the

MS/MS

spectrum

of

compound

Scheme

shows

how

this

ion

is

produced

from

compound

In

this

case

homolytic

cleavage

can

be

used

to

distribute

a

single

electron

onto

the

nitrogen

Compounds

that

contain

a

nitrogen

two

carbons

away

from

the

sulfation

site

like

this

one

could

also

produce

m/z

as

a

product

ion

Scheme

mechanism

producing

m/z

from

compound

Group

e

sulfated

amines

The

last

group

of

sulfated

products

sulfamates

originates

from

the

sulfation

of

amines

Compounds

belong

to

this

type

The

only

characteristic

ion

for

this

type

of

sulfated

products

is

the

negative

ion

m/z

This

characteristic

ion

can

be

obtained

from

fragmentation

of

sulfated

products

originating

from

both

aliphatic

amines

-LRB-

Fig.

-RRB-

and

aromatic

amines

-LRB-

Fig.

-RRB-

This

group

of

sulfated

products

has

another

type

of

bond

a

N-S

bond

that

has

a

small

electronegativity

difference

between

the

two

atoms

-LRB-

-RRB-

-LSB-

-RSB-

making

the

homolytic

cleavage

between

the

nitrogen

and

sulfur

favorable

The

mechanism

that

shows

production

of

m/z

is

illustrated

in

Scheme

No

other

fragmentation

pathway

is

as

favorable

as

this

homolytic

cleavage

pathway

making

m/z

the

predominant

ion

in

all

of

the

MS/MS

data

for

these

compounds

Scheme

amines

-LRB-

group

e

-RRB-

produce

the

characteristic

ion

m/z

Prediction

rules

For

each

type

of

sulfated

product

there

is

a

predominant

fragmentation

pathway

that

results

in

a

characteristic

product

ion

during

MS/MS

fragmentation

The

specific

characteristic

ion

for

each

group

is

obtained

from

every

sulfated

product

in

that

group

as

shown

in

Table

the

ion

with

the

neutral

loss

of

Da

can

be

seen

in

MS/MS

spectrum

of

every

sulfated

product

in

group

a

an

ion

with

m/z

can

be

detected

in

all

the

MS/MS

data

of

group

b

compounds

an

ion

with

m/z

can

be

obtained

from

MS/MS

data

of

all

the

sulfated

products

of

both

group

c

and

group

d

A

negative

ion

with

m/z

can

always

be

found

in

MS/MS

data

of

sulfated

products

in

group

e

We

have

shown

that

characteristic

fragmentation

pathways

of

sulfated

products

are

dependent

upon

the

structural

features

of

the

sulfation

sites

Conversely

relevant

structural

information

of

sulfation

sites

can

be

obtained

by

determining

characteristic

fragmentation

pathways

or

characteristic

ions

To

facilitate

this

information

transformation

a

set

of

prediction

rules

is

described

as

follows

If

the

characteristic

ion

with

a

neutral

loss

of

Da

can

be

detected

this

indicates

that

the

sulfate

group

is

attached

to

an

carbon

the

sulfated

product

is

therefore

produced

by

the

sulfation

of

a

phenol

or

an

enol.If

the

characteristic

ion

with

m/z

can

be

detected

the

sulfated

product

is

produced

by

sulfation

of

a

benzylic

or

allylic

alcohol

or

a

hydroxylamine.If

the

characteristic

ion

with

m/z

can

be

obtained

and

it

is

more

abundant

than

the

ion

m/z

-LRB-

if

it

is

detected

-RRB-

the

sulfate

group

is

attached

to

an

carbon

This

is

the

sulfated

product

produced

by

sulfation

of

aliphatic

alcohols

with

an

available

β

hydrogen

attached

to

an

carbon

If

of

the

ions

m/z

-LSB-

-RSB-

m/z

or

m/z

can

be

detected

and

only

the

ion

m/z

is

observed

then

the

sulfated

product

is

produced

by

sulfation

of

an

amine

There

are

occasions

when

the

conditions

in

rule

and

might

both

be

observed

For

example

both

of

these

apply

to

compound

which

suggests

that

the

sulfation

site

is

not

only

an

alcohol

with

available

β

hydrogen

-LRB-

on

an

carbon

-RRB-

but

also

a

benzylic

or

allylic

alcohol

and

this

estimation

is

consistent

with

the

actual

structure

of

compound

Among

the

selected

sulfated

products

of

them

follow

the

proposed

prediction

rules

The

exception

compound

possesses

an

amine

group

in

the

β

position

The

amine

at

this

position

enables

the

compound

undergo

the

fragmentation

pathway

to

produce

ion

with

m/z

-LRB-

see

Scheme

-RRB-

In

this

case

rule

incorrectly

predicts

the

sulfation

site

for

compound

As

a

result

the

obtained

data

in

Table

demonstrates

a

-LRB-

-RRB-

success

rate

of

these

prediction

rules

Potential

application

of

the

prediction

rules

in

characterizing

unknown

sulfated

metabolites

As

one

of

the

major

phase

II

biotransformations

sulfate

conjugation

is

involved

in

the

metabolism

of

an

enormous

range

of

substrates

-LSB-

-RSB-

In

general

sulfation

is

a

detoxification

or

deactivation

pathway

but

it

can

also

bioactivate

the

substrate

molecules

when

sulfation

sites

are

benzylic

allylic

alcohols

-LRB-

group

c

-RRB-

or

aromatic

hydroxylamines

-LRB-

group

d

-RRB-

-LSB-

-RSB-

According

to

the

prediction

rules

developed

herein

sulfation

at

any

of

these

sites

will

generate

products

that

produce

the

ion

m/z

in

MS/MS

so

the

structural

feature

of

the

sulfation

sites

can

be

determined

by

rule

Sulfation

of

other

sites

like

phenols

aliphatic

alcohols

or

amines

will

cause

other

characteristic

ions

to

appear

in

MS/MS

data

Therefore

the

sulfation

sites

can

be

determined

by

rule

or

In

these

cases

sulfation

facilitates

detoxification

With

the

proposed

prediction

rules

biological

effects

of

sulfation

can

be

determined

and

the

properties

of

sulfated

metabolites

can

be

estimated

Figure

demonstrates

how

the

proposed

method

works

to

characterize

unknown

sulfated

metabolites

Since

high-quality

MS/MS

data

is

a

necessary

prerequisite

of

using

this

method

if

the

metabolite

is

present

in

a

complex

biological

matrix

some

mass

spectral

optimization

may

be

necessary

After

obtaining

reliable

-LRB-

-RRB-

ESI-MS/MS

data

on

the

unknown

sulfated

metabolite

the

prediction

rules

are

used

to

determine

sulfation

site

by

identifying

the

characteristic

ions

present

in

the

MS/MS

data

If

the

MS/MS

data

matches

rule

or

-LRB-

instead

of

rule

-RRB-

the

sulfation

sites

can

be

phenols

-LRB-

or

enols

-RRB-

aliphatic

alcohols

-LRB-

with

β

hydrogen

on

carbon

-RRB-

or

amines

In

these

cases

the

sulfation

undergoes

a

detoxification

pathway

and

the

sulfated

metabolite

is

more

easily

eliminated

compared

to

the

parent

drug

If

the

MS/MS

data

is

consistent

with

rule

the

sulfation

site

should

be

benzylic

allylic

alcohols

or

hydroxylamines

This

would

indicate

that

the

sulfation

might

follow

a

bioactivation

pathway

and

lead

to

carcinogenic

sulfated

products

One

potential

caveat

to

this

second

condition

is

that

a

few

nontoxic

sulfated

metabolites

that

have

structures

similar

to

compound

might

also

produce

the

characteristic

ion

with

m/z

following

the

mechanism

in

Scheme

Another

possible

drawback

of

this

characterization

is

that

the

diagnostic

ion

m/z

does

not

discriminate

between

sulfated

products

of

aliphatic

and

aromatic

hydroxylamines

and

thus

far

only

aromatic

hydroxylamines

have

been

proven

to

be

toxic

However

distinguishing

between

aromatic

and

aliphatic

hydroxylamines

is

not

critically

important

because

sulfated

aliphatic

hydroxylamines

are

rather

uncommon

metabolites

While

it

is

possible

that

a

few

compounds

could

be

misassigned

as

toxic

of

the

sulfated

metabolites

that

are

known

to

be

bioactivated

would

be

misdiagnosed

as

nontoxic

as

demonstrated

herein

Fig.

method

for

characterizing

unknown

sulfated

metabolites

Conclusion

A

method

was

developed

to

determine

the

structural

features

of

sulfation

sites

by

detecting

the

characteristic

fragmentation

pathway

of

the

corresponding

sulfated

products

in

-LRB-

-RRB-

ESI-MS/MS

By

summarizing

MS/MS

data

from

five

different

types

of

sulfated

products

originating

from

different

sulfation

sites

their

characteristic

fragmentation

pathways

and

characteristic

ions

were

determined

Based

on

this

information

a

set

of

prediction

rules

was

developed

to

transfer

information

about

the

fragmentation

pathway

of

sulfated

products

to

the

structural

features

of

the

sulfation

site

As

a

result

the

proposed

prediction

rules

can

be

applied

in

drug

metabolite

profiling

to

characterize

sulfation

sites

to

further

estimate

the

biological

effect

of

sulfation

and

to

evaluate

relevant

properties

of

sulfated

metabolites

O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O B O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O B I O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O B O

mass spectrometry
pharmaceuticals
metabolites
sulfation

icp-ms
bioanalytical methods
biological samples


Application of at-line two-dimensional liquid chromatography -- mass spectrometry for identification of small hydrophilic angiotensin I-inhibiting peptides in milk hydrolysates A two-dimensional chromatographic method with mass spectrometric detection has been developed for identification of small , hydrophilic angiotensin I-inhibiting peptides in enzymatically hydrolysed milk proteins . The method involves the further separation of the poorly retained hydrophilic fraction from a standard C18 reversed-phase column on a hydrophilic interaction liquid chromatography -LRB- HILIC -RRB- column . The latter column is specifically designed for the separation of hydrophilic compounds . Narrow fractions collected from the HILIC column were analysed for their angiotensin I-converting enzyme -LRB- ACE -RRB- inhibiting potential in an at-line assay . Fractions showing significant inhibition of ACE were analysed by LC -- MS for structure elucidation . With this method the main peptides responsible for ACE-inhibition in the hydrophilic part of a milk hydrolysate could be determined . The ACE-inhibiting peptides RP , AP , VK , EK , and EW explained more than 85 % of ACE-inhibition by the hydrophilic fraction . Introduction Hypertension is one of the major health problems in the western world . A variety of food ingredients that help to reduce blood pressure have been described . Particularly attractive are hydrolysed proteins from fermented milk containing specific peptides that can inhibit the angiotensin I-converting enzyme -LRB- ACE -RRB- , an enzyme involved in blood-pressure regulation -LSB- 1 -- 3 -RSB- . For the development of optimized anti-hypertensive food products , it is crucial to know which specific peptide -LRB- s -RRB- in the complex protein hydrolysate is -LRB- are -RRB- responsible for the ACE-inhibition . Identification of active ingredients in complex mixtures is generally done using assay-guided repeated fractionation . For the specific case of ACE-inhibiting peptides in milk-protein hydrolysates often only part of the total activity of the product can be assigned to specific peptides -LSB- 4 -RSB- . A significant part of the remaining activity is present in fractions that are poorly retained by the standard reversed-phase columns used in the fractionation procedure . Nakamura et al. -LSB- 1 -RSB- for example could only explain part of the activity of a casein hydrolysate by the tri-peptides VPP and IPP eluting in one of the later fractions . The remaining activity was present in the unretained fraction . In our laboratory similar samples were seen to have over 30 % of the total activity in the unretained fraction . Identification of the oligopeptides in this fraction was not further pursued but would have been extremely difficult because the fraction contains far too many compounds for unambiguous activity assignment . Moreover , there are also too many interfering compounds present in the first-eluting fractions for reliable MS identification . Similar situations are also described by other authors -LSB- 5 -- 7 -RSB- . In a previous investigation we presented a new at-line method for the identification of ACE-inhibiting -LRB- oligo -RRB- peptides in hydrolysed caseinate -LSB- 4 -RSB- . Approximately 70 % of the activity of these samples was caused by peptides nicely retained by the two reversed-phase columns used in the fractionation . Twenty percent of the activity was present in the hydrophilic fraction . The remaining 10 % was caused by a large number of peptides with low ACE-inhibiting activity . Identification of the peptides in the hydrophilic fraction was impossible due to the large number of peptides present and the presence of co-eluting compounds , such as salts and sugars , that resulted in severe suppression of ionisation . Two reversed-phase columns were used in an at-line 2D set-up to provide improved resolution of the very complex mixture . Unfortunately , the highly hydrophilic peptides escaped identification since they were unretained on both columns . To identify the peptides responsible for the ACE inhibition of the unretained hydrophilic fraction improved chromatographic retention and separation is required . Hydrophilic interaction chromatography -LRB- HILIC -RRB- could be an attractive way of achieving this . The use of HILIC for separation of hydrophilic substances such as proteins , peptides , and nucleic acids was first described by Alpert in 1990 -LSB- 8 -RSB- . Since then HILIC has been used for the separation of many different substances , for example denaturants -LSB- 9 -RSB- , polar oligomers -LSB- 10 -RSB- , and pharmaceutical ingredients -LSB- 11 -RSB- . Many publications can be found describing the increased retention of hydrophilic peptides on HILIC columns in comparison with reversed-phase columns . A detailed review is published by Yoshida -LSB- 12 -RSB- . Literature describing the application of reversed-phase separation in combination with HILIC chromatography is scarce . A notable exception is the compositional analysis of an enzymatic protein hydrolysate obtained from acid-deaminated wheat gluten by Schlichtherie-Cerny -LSB- 13 -RSB- . The hydrolysate was first fractionated by GPC . The fraction corresponding to a molecular weight range of 100 -- 750Da was then further fractionated by RPLC . Finally , the peptides in the unretained peak from the RPLC column were separated and characterised by HILIC -- API-MS . Using this method ten amino acids , eight di-peptides and one tri-peptide , were identified in this fraction . In this paper we describe the development of a method for the identification of ACE inhibiting peptides in the poorly retained fractions of a reversed-phase fractionation of an enzymatically hydrolysed milk protein . The possibility of improving chromatographic resolution by using a HILIC separation as a second-dimension separation after the first reversed-phase isolation is studied . The aim of the new method is to extend the applicability of a previously developed system for the identification of ACE-inhibiting peptides in complex peptide mixtures such as protein hydrolysates -LSB- 4 -RSB- . The applicability of the method is demonstrated by the identification of hydrophilic ACE-inhibiting peptides in an enzymatically hydrolysed milk protein . Experimental Chemicals The model peptides VPP , IPP , and LPP -LRB- purity > 98 % -RRB- were purchased from Bachum -LRB- Dübendorf , Switzerland -RRB- . All other peptides were synthesised by JPT Peptide Technologies -LRB- Berlin , Germany -RRB- . For HPLC analysis , acetonitrile , formic acid , and trifluoroacetic acid were purchased from Merck -LRB- Amsterdam , The Netherlands -RRB- . Aqueous ammonia -LRB- Merck -RRB- was used for neutralization of the HPLC fractions . For instrument tuning PEG 300 , PEG 600 , and PEG 1000 were purchased from Sigma Chemicals -LRB- St Louis , MO , USA -RRB- . Ammonium acetate was obtained from Merck . For the at-line assay angiotensin I-converting-enzyme -LRB- ACE -RRB- and hippuryl -- histidyl -- leucine -LRB- HHL -RRB- were purchased from Sigma -- Aldrich Chemie -LRB- Zwijndrecht , The Netherlands -RRB- . PBS buffer was purchased from Gibco -LRB- Paisley , UK -RRB- . The hydrolysed milk powder was an enzymatically hydrolysed milk protein obtained from Calpis -LRB- Tokyo , Japan -RRB- . Instrumentation All peptide separations and identifications were performed with a Waters Alliance 2795 HT HPLC coupled to a Micromass QTOF-Ultima hybrid time-of-flight mass spectrometer equipped with a lock spray option for accurate mass determination -LRB- Waters , Almere , The Netherlands -RRB- . Fractions were collected in polypropylene 300-μL 96-well plates -LRB- Nunc , Roskilde , Denmark -RRB- using a Mark IV fraction collector also from Waters . Solvent evaporation was performed using an Ultravap 96-well evaporation device -LRB- Porvair , Shepperton , UK -RRB- . Quantification of HHL and HL in the at-line assay was performed on the QTOF instrument described above . Two-dimensional analytical separation The first-dimension reversed-phase separation was performed on two serially connected 150 × 2.1 mm Inertsil 5 ODS3 columns , particle size 5μm , operating in the gradient mode -LRB- Varian , Middelburg , The Netherlands -RRB- . The guard column was a 20 × 3.9 mm SymmetryShield RP8 column packed with 5-μm particles -LRB- Waters , Etten-Leur , The Netherlands -RRB- . Mobile phase A consisted of 0.1 % TFA in Milli-Q water . Mobile phase B consisted of 0.1 % TFA in acetonitrile . The initial eluent composition was 100 % A . After a 5 min hold a linear gradient was started to 5 % B in 15 min , followed by a linear gradient to 70 % B in 75 min . Finally , mobile phase composition was programmed to 99 % B in 1 min and kept at 99 % B for another 4 min . At the end of the gradient the eluent composition was reduced to 100 % A in 6 min and the system was allowed to re-equilibrate for 14 min . The total run time was 120 min . The eluent flow rate was 0.2 mL min − 1 and the column temperature was 60 °C . In-house experiments revealed that at this temperature isomeric peptides such as IPP and LPP are separated . UV detection was performed at 215nm and 254nm . Fractions of 200μL were collected in 96-well plates . After neutralization and solvent evaporation the residues were reconstituted in 50μL of an 80:20 mixture of solvents A and B for the second-dimension HILIC analysis . The second dimension HILIC analysis was performed on a 150 × 2.1 mm HILIC Atlantis column with a particle size of 3μm -LRB- Waters , Etten-Leur , The Netherlands -RRB- . Here solvent A consisted of 0.1 % formic acid in acetonitrile and solvent B of 10mmol L − 1 aqueous ammonium acetate solution +0.1 % formic acid in Milli-Q water . The separation was performed at 40 °C , the maximum temperature specified by the manufacturer , and a flow rate of 0.2 mL min − 1 . The initial eluent composition was 95 % A . A linear gradient was used to 70 % B in 30 min , followed by a linear gradient to 90 % B in 5 min where it was kept for another 5 min . At the end of the run the eluent was reduced to 95 % C in 2 min . The eluent flow was then increased to 0.3 mL min − 1 in 1 min . This flow rate was maintained for another 7 min . Finally the flow was reduced to 0.2 mL min − 1 in 1 min . and the system was allowed to equilibrate for 3 min . The total run time was 54 min . 45μL of each fraction collected from the ODS3 column was injected . At the outlet of the second dimension HILIC column fractions of 200μL were collected in 96-well plates . The fractions were neutralized , evaporated , and transferred to the activity assay -LRB- next section -RRB- . At-line Matsui assay ACE inhibition by the collected fractions was measured in a 96-well plate assay according to the at-line method described in an earlier publication -LSB- 1 -RSB- . The method is based on the conversion of hippuryl -- histidyl -- leucine -LRB- HHL -RRB- into hippurate -LRB- H -RRB- and histidyl -- leucine -LRB- HL -RRB- by the angiotensin I-converting enzyme -LRB- ACE -RRB- . Briefly , after evaporation of the eluent the fractions were reconstituted in 50μL PBS buffer and vortex mixed for 1 min . This solution -LRB- 40μL -RRB- was used for the at-line assay . Percentage inhibition was calculated from the peak areas of HHL and H at 280nm . For the active fractions the remaining 10μL was used for identification of the peptides . Structural identification of ACEI peptides Peptide identification was performed on the QTOF MS instrument using HPLC -- MS and MS -- MS in the accurate mass mode . Fractions collected after the second dimension that showed activity were separated on the HILIC column now coupled to the MS using the gradient described above . The source and desolvation temperatures were 100 °C and 300 °C , respectively . The cone and desolvation gas flows were 100L h − 1 and 800L h − 1 , respectively . The capillary voltage was 4kV and the cone voltage 35V . The collision energy was 10eV in full-scan mode and 25eV in MS -- MS mode . The collision gas was argon and the analyser pressure was 4 × 10 − 5 mbar . In the lock spray probe a mixture of PEG 300 , PEG 600 , and PEG 1000 dissolved in 10mmol L − 1 ammonium acetate in methanol -- water 1:1 -LRB- v/v -RRB- at concentrations of 0.01 , 0.02 , and 0.04 μg mL − 1 , respectively , was used . The reference flow was 5μL min − 1 . Spectra were recorded at a resolution of 10,000 . Masslynx software -LRB- Waters -RRB- was used for data acquisition and evaluation . Results and discussion In a reversed-phase fractionation of a protein hydrolysate a significant percentage of the activity can be present in the unretained hydrophilic fraction . To be able to calculate the contribution of this fraction of our milk hydrolysate to the total activity , an ACE-inhibition profile of the entire product was produced . To this end 20μL of a 20 mg mL − 1 solution of the milk hydrolysate in solvent A was injected on to the 300 mm ODS3 column . Fractions of 200μL each were collected from 0 to 65 min , with an interval time of 1 min , in a 96 well plate . The ACE inhibition of each fraction was determined . Figure 1a shows the MS TIC of the fermented milk and Fig. 1b shows the activity profile of the same analysis . From the data in Fig. 1b it was calculated that the hydrophilic compounds in fractions 6 to 15 represent approximately 25 % of the total ACE inhibition . MS -- MS showed that the high activity in fractions 27 to 31 resulted from the known ACE inhibiting peptides IPP and LPP . The activity in fractions 21 to 23 resulted from the peptide VPP . These peptides have been identified to be the main contributors to the ACE-inhibition of a Lactobacillus helveticus-fermented milk -LSB- 2 -RSB- and represent 40 % of the total ACE inhibition . The remaining activity between fractions 16 and 40 is most likely caused by a mixture of ACE-inhibiting peptides of different chain lengths . This is still under investigation . In the region from fraction 6 to fraction 15 a large number of peptides co-elute and the spectra of the fractions are highly complex . Figure 2 , for example , shows the mass spectrum of fraction 7 collected from the first dimension ODS 3 columns and the ion traces of three of the ACE-inhibiting peptides discussed later in this manuscript . At least 16 ions can be observed with intensities above 10 % of the base peak , all representing different peptides . In order to be able to assign the measured activity to one or more of these peptides a further separation in an additional chromatographic dimension is required . To achieve this , fractions collected from the first-dimension ODS column were injected on the second dimension HILIC column . Again 200-μL fractions were collected in 96-well plates . After neutralization and evaporation the residues were reconstituted in 50μL Milli-Q water . Of this , 40μL was used for ACE inhibition testing , the remaining 10μL for MS identification . As an example of the results of these experiments , Fig. 3 shows the TIC chromatogram obtained from full-scanning analysis of fraction 7 of the ODS3 columns , now separated on the second dimension HILIC column . The TIC chromatogram clearly shows that the composition of this fraction is indeed very complex . To identify the peptide -LRB- s -RRB- responsible for the ACE-inhibition , the fractions collected in the 96-well plate were subjected to the ACE-inhibition assay . Figure 4 displays the activity distribution of ODS fraction 7 . The data in Fig. 4 are corrected for the background recorded using a blank gradient , i.e. a gradient without injection . Blank analysis of the HILIC column showed slightly negative values compared with a reference of pure PBS . The reason for these negative responses is still unknown . The results in Fig. 4 clearly show the strong activity in HILIC fractions 17 to 19 and somewhat lower activity in fractions 12 to 16 from the HILIC column . LC -- MS separations were performed on these fractions and the MS data were searched for peptides in the two active regions . In Fig. 5 the mass spectrum of fraction 18 is given . In the spectrum three abundant ions can be observed . A library search and MS -- MS measurements revealed that m/z 272.1721 represents the molecular ion of the di-peptide RP -LRB- − 0.7 ppm -RRB- , while m/z 253.1190 represents the molecular ion of HP -LRB- − 2.1 ppm -RRB- and m/z 235.1305 the loss of water from m/z 253.1190 . Identification of the peptides in the remaining fractions of the HILIC column showed that fraction 7 of the first-dimension ODS3 column consisted of three free amino acids , 19 di-peptides and 2 tri-peptides while six compounds remained unidentified . The positively identified amino acids were E , Q , and K and the di-peptides were QD , KY , ER , RE , KP , HP , RP , AP , VK , EK , EW , and PH. The di-peptides ET , TP , TQ , PQ , KV , KE , and HK and the tri-peptides APK and VRG were tentatively identified . Structure confirmation was based on elution time , measured exact mass -LRB- error < 5ppm -RRB- and MS -- MS fragmentation pattern compared with those of the model compounds . Using this method the remaining hydrophilic fractions 5 to 15 collected from the ODS3 column were also analysed in the 2D mode . In Fig. 6 a three-dimensional plot of the activity distribution over the fractions of the two columns is given . In total , five amino acids , 35 di-peptides , 13 tri-peptides , one penta-peptide and 18 not yet identified compounds were found . The identities of all amino acids and the sequence of 27 di-peptides were again confirmed by use of model compounds . Table 1 lists the sequences of the identified peptides together with their reported IC50 values and ACE inhibition data at 20μmol L − 1 established in house -LRB- Foltz et al. manuscript in preparation -RRB- . For peptides for which no model compounds were available , identification was based solely on the fragmentation pattern in MS -- MS. Fig. 1LC separation of a 20 mg mL − 1 solution of the milk hydrolysate powder on the ODS-3 reversed-phase column . a MS-TIC chromatogram . b Activity profile . Error bars indicate the standard deviation -LRB- n = 3 -RRB- . Fraction volume 200 μLFig . 2Mass spectrum of fraction 7 collected from the first-dimension ODS3 columnFig . 3MS-TIC of fraction 7 from the ODS3 column analysed on the HILIC column . The inset shows the selected ion traces of three of the most active peptidesFig . 4ACEI profile of fraction 7 from the ODS3 column analysed on the HILIC columnFig . 5Mass spectrum of fraction 18 collected from the HILIC columnFig . 6Three-dimensional display of the ACEI distribution of the fractions collected from the ODS3 column and the HILIC columnTable 1Peptides identified in fractions 6 to 15 of the ODS3 column , analysed on the HILIC columnelution -LRB- min -RRB- timepeptide sequenceIC50 value -LRB- μM -RRB- literatureLiterature referenceInhibition at 20 μM -LRB- % -RRB- peak area -LRB- counts -RRB- elution time -LRB- min -RRB- peptide sequencevalue -LRB- μM -RRB- literatureLiterature referenceInhibition 20 μM -LRB- % -RRB- peak area -LRB- counts -RRB- 11.64 Fn.r . 8316.34 FP1215 .713 n.m. 63111.90 Yn.r . 227416.41 HSMn.r . 65612.02 EIn.r . 38.2222116.43 EPFn.r . 18612.36 IEn.r . 3.7459116.43 DKIn.r . 18712.40 YP72013n.m .5816.52 KY1 .63,7.8,1316,13,17 n.m. 1312.59 EVn.r . 25.462216.66 VYP28813 12512.59 VEn.r . 41.02217.00 EWn.r . 65.1157812.59 IP1291317 .710017.22 VK12 .91357.216712.84 VP5751345 .980117.23 ERn.r . n.m. 23613.21 En.r . n.m. 28817.30 SGYn.r . 62413.26 TEn.r . 26.034417.35 MEn.r . 24.64213.44 LQn.r . 11.274817.55 Kn.r . n.m. 49713.74 KP16 ,22,3013,1533.714417.61 EKn.r . 41.833713.81 VNEn.r . 137817.84 REn.r . n.m. 16614.08 LNn.r . 14.727117.86 KEn.r . 13.48314.10 Qn.r . n.m. 51117.89 KV33 n.m. 23514.10 QDn.r . n.m. 79118.04 VVRn.r . 11414.28 TDVENn.r . 201918.08 HPn.r . 11.1142714.50 PTn.r . 3.1100118.18 KVPn.r . 4514.56 NVPn.r . 274418.24 VPQn.r . 50514.61 YQn.r . n.m. 3618.63 RP21 ,91,1821336.4134014.64 SPPn.r . 33518.97 PHn.r . 0.030614.72 TQn.r . 0.017519.03 QPn.r . n.m. 40214.86 AHn.r . 7.310119.11 KP16 ,22,3013,1533.614414.91 PP2284 .7180.0175919.22 APKn.r . n.m. 26615.01 AP29 ,26913,1011.481120.18 VRGn.r . n.m. 31315.18 PQn.r . 0.0104120.47 HKn.r . 0.0398 The sequences of the bold printed peptides were confirmed by MS -- MS analyses of reference peptides , n.r. , not reported in the literature ; n.m. , not measured To verify whether the identified peptides indeed fully explain the measured activity of the hydrophilic fraction the concentration of all peptides should be determined . Next a mixture should be prepared which is then analysed using the two-dimensional method described in this article . The ACE-inhibition profile of this mixture should be identical with that of the sample . Such an experiment will hardly be feasible because it requires the availability of all the components . To estimate of the completeness of identification , the contribution of each peptide was estimated from the measured peak area and the IC50 value obtained from literature or from unpublished in-house data . To estimate the contribution of an individual peptide the following relationship was used : CA ≈ Ap/IC50p . In this equation CA represents the contribution of the peptide to the measured ACE-inhibition , Ap the peak area , and IC50p the IC50 value of the peptide . Clearly this proportionality equation is only approximate as it assumes that the MS response for all peptides is identical . When both the peak area and the IC50 value are taken into consideration it can be concluded that the di-peptides AP , RP , VK , and EK are the main peptides responsible for the measured ACE-inhibition of the hydrophilic fractions 6 to 10 collected from the ODS3 column -LRB- Fig. 7 -RRB- . In Fig. 8 the activities of all peptides in each fraction are summarized and the profile is compared with that of the measured profile . The two profiles are similar . The differences between the graphs can be explained from the fact that no correction was applied for differences in MS responses of the various peptides . Fig. 7Distribution of the ACE-inhibiting activity , calculated from Table 1 , in hydrophilic fractions 5 to 10 collected from the ODS3 columnFig . 8Calculated -LRB- bottom -RRB- and measured -LRB- top -RRB- ACE inhibition profiles The remaining fractions 11 to 15 of the ODS3 column were analysed in the same way as fractions 6 to 10 . These analyses showed that the peptide EW is responsible for the major part of the ACE-inhibition measured in these fractions . In total , the five peptides identified in the fractions 6 to 15 are responsible for approximately 85 % of the activity measured in the hydrophilic fractions . The most important contribution is that of the di-peptide RP , which is responsible for 34 % . Together with the peptides VPP , IPP , and LPP now approximately 65 % of the total activity of the product has been explained . The remaining 35 % is distributed over a large number of peptides with relatively low overall activities . When compiling the list of published IC50 values for the peptides identified in this study in some cases large differences were seen for the IC50 values of proline-containing peptides in different publications . The reported IC50 values for AP , for example , vary between 29 and 269 μmol L − 1 -LSB- 14 -RSB- . This large difference might be caused by variations in the cis-to-trans ratio of the proline present in synthetic model compounds as a result of differences in the synthetic routes -LRB- Fmoc or Boc -RRB- , as was shown for the peptide DKIHP by Gómez-Ruiz -LSB- 15 -RSB- . In our calculations the trans-Pro value of 29 μmol L − 1 for AP was used , because trans-Pro is known to be dominant in natural products -LSB- 15 -RSB- . Many of the identified di-peptides were found in milk hydrolysates for the first time . For most of the newly identified ACE active peptides it is actually the first time they are reported at all . The long list of newly identified peptides found here clearly demonstrates the potential of the two dimensional separation approach of HILIC and reversed-phase HPLC described here . Conclusions Two-dimensional liquid chromatography in combination with mass spectrometry was successfully used for identification of poorly retained peptides present in enzymatically hydrolysed milk protein . A standard C18 reversed-phase column was used for the first separation followed by a second dimensional separation on a HILIC column . This two-dimensional procedure significantly improves the separation of hydrophilic peptides that elute almost unretained on a reversed-phase column and co-elute with numerous other compounds such as carbohydrates and salts . These compounds suppress the MS ionization and complicate identification . The method enabled the identification of hydrophilic peptides in complex mixtures . In the hydrophilic fraction of the milk hydrolysate investigated 71 compounds were found , including five free amino acids , 35 di-peptides , 12 tri-peptides , one penta-peptide , and 18 not yet identified compounds . Five peptides , RP , AP , VK , EK , and EW were responsible for approximately 85 % of the measured activity of the hydrophilic fraction ; of these RP made the highest contribution of 34 % . None of the five peptides has , to our knowledge , been reported earlier in milk hydrolysates or related products such as yogurt or cheese . Most of the peptides involved remained undetected in single-dimensional chromatography .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O B O B O B O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

angiotensin-converting enzyme
blood pressure-lowering peptides
reversed-phase