Journal of Chromatography Open 5 (2024) 100134
Available online 10 May 2024
2772-3917/© 2024 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC license (
http://creativecommons.org/licenses/by-
nc/4.0/
).
Enantioselective
three-dimensional
high-performance
liquid
chromatographic
determination
of amino
acids
in the
Hayabusa2
returned
samples
from
the
asteroid
Ryugu
Aogu
Furusho
a
,
b
, Chiharu
Ishii
a
, Takeyuki
Akita
a
, Mai
Oyaide
a
, Masashi
Mita
c
,
Hiroshi
Naraoka
d
, Yoshinori
Takano
e
, Jason
P. Dworkin
f
, Yasuhiro
Oba
g
, Toshiki
Koga
e
,
Kazuhiko
Fukushima
h
, Dan
Aoki
h
, Minako
Hashiguchi
i
, Hajime
Mita
j
, Yoshito
Chikaraishi
g
,
Naohiko
Ohkouchi
e
, Nanako
O.
Ogawa
e
, Saburo
Sakai
e
, Daniel
P. Glavin
f
, Jamie
E. Elsila
f
,
Eric
T. Parker
f
, Jos
́
e C. Aponte
f
, Hannah
L. McLain
f
, Francois-Regis
Orthous-Daunay
k
,
V
́
eronique
Vuitton
k
, Roland
Thissen
l
, C
́
edric
Wolters
k
, Philippe
Schmitt-Kopplin
m
,
Alexander
Ruf
n
, Junko
Isa
o
, Norbert
Hertkorn
m
, John
M.
Eiler
p
, Toshihiro
Yoshimura
e
,
Haruna
Sugahara
q
, Heather
V. Graham
e
, Yoshihiro
Furukawa
r
, Daisuke
Araoka
s
,
Satoru
Tanaka
t
, Takaaki
Yoshikawa
u
, Fumie
Kabashima
v
, Kazunori
Sasaki
w
, Hajime
Sato
w
,
Tomoya
Yamazaki
g
, Morihiko
Onose
t
, Mayu
Morita
t
, Yuki
Kimura
g
, Kuniyuki
Kano
x
,
Junken
Aoki
x
, Kosuke
Fujishima
o
, Shin-ichiro
Nomura
y
, Shogo
Tachibana
q
,
z
,
Tomoki
Nakamura
r
, Takaaki
Noguchi
aa
, Ryuji
Okazaki
d
, Hikaru
Yabuta
ab
,
Hisayoshi
Yurimoto
ac
, Kanako
Sakamoto
q
, Toru
Yada
q
, Masahiro
Nishimura
q
, Aiko
Nakato
q
,
ad
,
Akiko
Miyazaki
q
, Kasumi
Yogata
q
, Masanao
Abe
q
,
ae
, Tomohiro
Usui
q
, Makoto
Yoshikawa
q
,
ae
,
Takanao
Saiki
q
, Satoshi
Tanaka
q
,
ae
, Fuyuto
Terui
af
, Satoru
Nakazawa
q
, Tatsuaki
Okada
q
,
ag
,
Sei-ichiro
Watanabe
i
, Yuichi
Tsuda
q
, Kenji
Hamase
a
,
*
a
Graduate
School
of Pharmaceutical
Sciences,
Kyushu
University,
Fukuoka
812-8582,
Japan
b
School
of Pharmaceutical
Sciences,
University
of Shizuoka,
Shizuoka
422-8526,
Japan
c
KAGAMI,
Inc.,
Osaka
567-0085,
Japan
d
Department
of Earth
and
Planetary
Sciences,
Kyushu
University,
Fukuoka
819-0395,
Japan
e
Biogeochemistry
Research
Center,
Japan
Agency
for Marine-Earth
Science
and
Technology,
Yokosuka
237-0061,
Japan
f
Solar
System
Exploration
Division,
NASA
Goddard
Space
Flight
Center,
Greenbelt,
MD 20771,
United
States
g
Institute
of Low
Temperature
Science,
Hokkaido
University,
Sapporo
060-0189,
Japan
h
Graduate
School
of Bioagricultural
Sciences,
Nagoya
University,
Nagoya
464-8601,
Japan
i
Graduate
School
of Environmental
Studies,
Nagoya
University,
Nagoya
464-8601,
Japan
j
Department
of Life,
Environment
and
Applied
Chemistry,
Fukuoka
Institute
of Technology,
Fukuoka
811-0295,
Japan
k
Institut
de Planetologie
et d
’
Astrophysique
de Grenoble,
University
Grenoble
Alpes,
38000
Grenoble,
France
l
Institut
Chimie
Physique,
CNRS,
University
of Paris-Saclay,
91405
Orsay,
France
m
Analytical
BioGeoChemistry,
Helmholtz
Munich,
85764
Neuherberg,
Germany
n
Laboratoire
de Physique
des Interactions
Ioniques
et Mol
́
eculaires,
Universit
́
e Aix-Marseille,
13397
Marseille,
France
o
Earth-Life
Science
Institute,
Tokyo
Institute
of Technology,
Tokyo
152-8550,
Japan
p
Division
of Geological
and
Planetary
Sciences,
California
Institute
of Technology,
Pasadena,
CA 91125,
United
States
q
Institute
of Space
and
Astronautical
Science,
Japan
Aerospace
Exploration
Agency
(JAXA),
Sagamihara
252-5210,
Japan
r
Department
of Earth
Science,
Tohoku
University,
Sendai
980-8578,
Japan
s
Geological
Survey
of Japan,
National
Institute
of Advanced
Industrial
Science
and
Technology,
Tsukuba
305-8567,
Japan
t
Horiba
Techno
Service
Co.,
Ltd.,
Kyoto
601-8510,
Japan
u
Horiba
Advanced
Techno,
Co.,
Ltd.,
Kyoto
601-8510,
Japan
v
LECO
Japan
Corporation,
Tokyo
105-0014,
Japan
w
Human
Metabolome
Technologies,
Inc.,
Tsuruoka
997-0052,
Japan
x
Graduate
School
of Pharmaceutical
Sciences,
University
of Tokyo,
Tokyo
113-0033,
Japan
* Corresponding
author.
E-mail
address:
hamase@phar.kyushu-u.ac.jp
(K.
Hamase).
Contents
lists
available
at ScienceDirect
Journal
of Chromatography
Open
journal
homep
age:
www.elsevi
er.com/loc
ate/jcoa
https://doi.org/10.1016/j.jcoa.2024.100134
Received
18
February
2024;
Received
in revised
form
24
April
2024;
Accepted
25
April
2024
Journal of Chromatography Open 5 (2024) 100134
2
y
Department
of Robotics,
Tohoku
University,
Sendai
980-8579,
Japan
z
Department
of Earth
and
Planetary
Science,
University
of Tokyo,
Tokyo
113-0033,
Japan
aa
Division
of Earth
and
Planetary
Sciences,
Kyoto
University,
Kyoto
606-8502,
Japan
ab
Department
of Earth
and
Planetary
Systems
Science,
Hiroshima
University,
Higashi-Hiroshima
739-8526,
Japan
ac
Department
of Earth
and
Planetary
Sciences,
Hokkaido
University,
Sapporo
060-0810,
Japan
ad
Antarctic
Meteorite
Research
Center,
National
Institute
of Polar
Research,
Tachikawa
190-8518,
Japan
ae
School
of Physical
Sciences,
The
Graduate
University
for Advanced
Studies,
Hayama
240-0193,
Japan
af
Department
of Mechanical
Engineering,
Kanagawa
Institute
of Technology,
Atsugi
243-0292,
Japan
ag
Department
of Chemistry,
University
of Tokyo,
Tokyo
113-0033,
Japan
ARTICLE
INFO
Keywords:
Amino
acids
Enantiomer
separation
Three-dimensional
HPLC
Meteorite
Asteroid
Hayabusa2
ABSTRACT
The
chirality
of amino
acids
in extraterrestrial
materials
may
provide
an
insight
into
the
origin
of the
essential
L
-
enantiopure
amino
acids
in the
terrestrial
biosphere.
In 2020,
the
Hayabusa2
mission
succeeded
in bringing
back
surface
materials
from
the
C-type
asteroid
(162173)
Ryugu
to the
Earth.
Amino
acids
were
one
of the
targeted
organic
molecules
to be
studied
in the
Ryugu
samples.
To
analyze
the
various
structural
isomers
of amino
acids,
which
were
expected
to be
present,
from
the
limited
amount
of the
returned
samples,
the
development
of a
highly-sensitive
and
selective
analytical
method
was
necessary.
In the
present
study,
a three-dimensional
high-
performance
liquid
chromatography
(3D-HPLC)
system
has
been
developed
for
the
enantioselective
determi
-
nation
of five
proteinogenic
and
three
non-proteinogenic
amino
acids
in the
Ryugu
samples,
in which
amino
acids
in the
sample
were
separated
by
reversed-phase,
anion-exchange
and
enantioselective
columns
after
the
fluorescence
derivatization
with
4-fluoro-7-nitro-2,1,3-benzoxadiozole.
The
applicability
of the
analytical
system
to
the
extraterrestrial
samples
was
evaluated
by
analyzing
several
types
of
carbonaceous
meteorites
before
applying
the
system
to the
Ryugu
samples.
In the
analysis
of the
Ryugu
samples,
all
of the
target
amino
acids
were
successfully
determined
quantitatively.
Non-proteinogenic
amino
acids
including
2-amino-
n
-butyric
acid,
isovaline
and
norvaline,
rarely
present
in the
terrestrial
environment,
were
found
as almost
racemic
mixtures
with
47.1
to 55.2%
L
.
1. Introduction
Extraterrestrial
materials
containing
organic
compounds
provide
clues
to
elucidate
the
origin
and
evolution
of
the
Solar
System,
the
surface
environment
of the
early
Earth
and
perhaps
the
origin
of life
on
the
Earth.
Organic
analyses
of the
Murchison
carbonaceous
meteorite,
which
fell
in 1969,
showed
that
the
meteorite
contained
a variety
of
amino
acids
up
to ~100
[1].
Their
carbon
isotopic
ratios
were
different
from
those
on
the
Earth
[2,3],
and
many
non-proteinogenic
amino
acids
rarely
present
in the
terrestrial
environment
were
observed
[4,5],
sug
-
gesting
an
extraterrestrial
origin.
Excesses
of
L
-enantiomers
to
their
counterparts,
the
D
-enantiomers,
have
been
reported
for
some
amino
acids
in
the
Murchison
meteorite
[6
–
12].
Similar
L
-enantiomeric
ex
-
cesses
have
also
been
found
in other
meteorites,
such
as Murray
[8
–
10],
Orgueil
[13]
and
Tagish
Lake
[14].
The
terrestrial
biosphere
is over
-
whelmingly
dominated
by
the
L
-enantiomers
of amino
acids
both
as the
components
of proteins
and
biochemical
intermediates
with
the
D
-en
-
antiomers
generally
found
in
microbes
[15,16].
Several
hypotheses
about
the
origin
of the
homochirality
of terrestrial
amino
acids
have
been
proposed;
however,
they
are
still
under
discussion
[17,18].
Amino
acids
in meteorites,
asteroids
and
comets
are
thought
to be
one
of the
possible
source
materials
for
the
origin
of life
on
the
Earth,
and
the
enantiomeric
excesses
of meteoritic
organic
compounds
may
have
been
responsible
for
breaking
the
chiral
symmetry
on
the
early
Earth
[19].
Since
L
-amino
acids
are
rich
in the
present
terrestrial
environment,
the
contamination
of
L
-amino
acids
is always
a concern
in the
analyses
of
meteorites
collected
on
the
Earth.
Thus,
several
sample
return
missions
have
been
carried
out
to collect
and
bring
back
samples
from
primitive
small
bodies
such
as asteroids
and
comets.
The
National
Aeronautics
and
Space
Administration
(NASA)
obtained
samples
from
comet
Wild
2 by
the
Stardust
spacecraft
[20],
and
the
Japan
Aerospace
Exploration
Agency
(JAXA)
executed
the
Hayabusa
mission
to collect
samples
from
the
asteroid
Itokawa
[21].
These
projects
resulted
in
the
finding
of
glycine
(Gly)
and
several
non-proteinogenic
amino
acids
[22
–
24].
JAXA
next
launched
the
Hayabusa2
spacecraft
to
the
asteroid
Ryugu
on
December
3, 2014.
After
two
touchdown
samplings
on
February
21,
2019
(sample
A)
and
July
11,
2019
(sample
C),
the
Hayabusa2
space
-
craft
came
back
to the
Earth
with
5.4
g of Ryugu
samples
on December
6,
2020
[25].
Ryugu
is categorized
as a C
b
-type
asteroid,
whose
chemical
and
mineralogical
characteristics
are
very
similar
to
the
primitive
Ivuna-type
(CI)
carbonaceous
meteorite,
containing
more
organic
car
-
bon
than
the
S-type
asteroids
like
Itokawa
[26].
Therefore,
the
Ryugu
sample
was
expected
to provide
information
especially
about
organic
matters
including
amino
acids
in the
primitive
Solar
System,
which
may
be
related
to the
origin
of life
on
the
Earth.
Though
the
return
of 5.4
g of material
from
Ryugu
is remarkable
and
far
more
than
required
for
mission
success,
the
sample
is still
rare
and
immensely
valuable.
Since
the
identification
of specific
amino
acids
is a
destructive
analytical
process
and
the
most
similar
class
of meteorites
(CI)
shows
a very
complex
distribution
of
organic
compounds,
an
analytical
technique
with
both
high
sensitivity
and
selectivity
is
required
for
the
precise
determination
of amino
acid
enantiomers
in the
limited
amount
of
the
Ryugu
samples.
For
the
Ryugu
samples,
the
detection
of 23
proteinogenic
and
non-proteinogenic
amino
acids
were
reported
by
high-performance
liquid
chromatography
(HPLC)
using
a
HILIC
column
combined
with
mass
spectrometry
(MS),
in
which
no
chiral
separation
of the
amino
acids
was
achieved
nor
their
concentra
-
tions
determined
[27].
Several
methods
using
gas
chromatography
(GC)
[28
–
31]
and
liquid
chromatography
(LC)
[31
–
33]
have
so far
been
developed
and
utilized
for
the
enantioselective
analysis
of amino
acids
in extraterrestrial
sam
-
ples.
As
for
the
GC
method,
an
enantioselective
capillary
column
including
Chirasil-Val
or
Chirasil-Dex
was
utilized,
and
MS
was
frequently
equipped
to detect
amino
acids
derivatized
with
non-chiral
reagents
such
as
trifluoroacetic
anhydride/isopropanol
[29,34,35].
A
reversed-phase
LC
method
has
also
been
developed
for
extraterrestrial
amino
acids
using
chiral
derivatization
with
o
-phthaldialdehyde
(OPA)
plus
N
-acetyl-
L
-cysteine
(NAC)
[31,36].
By
using
these
methods,
a va
-
riety
of amino
acids
has
been
found
in meteorite
samples,
and
their
chiral
properties
have
also
been
obtained.
Most
of these
methods
were
designed
to
use
one
separation
column
equipped
with
fluorescence
detection
(FD)
and/or
MS.
Such
an LC
method
equipped
with
FD
and
MS
detection
have
successfully
been
applied
to the
Ryugu
samples
to detect
enantiomeric
amino
acids
[37,38].
However,
the
chromatographic
separations
obtained
by
the
one-dimensional
methods
were
sometimes
insufficient,
and
the
interfering
compounds
disturbed
the
determination
A. Furusho
et al.
Journal of Chromatography Open 5 (2024) 100134
3
of
trace
amounts
of
the
amino
acid
enantiomers,
and
the
isobaric
structural
isomers
were
also
usually
difficult
to be distinguished
also
by
MS
[13].
To
achieve
the
highly-selective
and
sensitive
determination
of chiral
amino
acids,
multi-dimensional
HPLC
is one
of the
most
suitable
ap
-
proaches
[39
–
45]. The
three-dimensional
(3D)
HPLC
system
[43
–
45] is
especially
promising,
in which
amino
acids
in the
sample
are
derivatized
with
a fluorescence
reagent
for
the
highly-sensitive
detection
followed
by
three
different
separation
modes
integrating
reversed-phase,
anio
-
n-exchange
and
enantioselective
columns.
In a previous
study
[37], we
performed
the
initial
analysis
of Hayabusa
2 mission,
and
reported
the
occurrence
of six
amino
acids
in the
Ryugu
sample
(A0106).
In the
initial
analysis,
the
analyzed
and
found
amino
acids
were
alanine
(Ala),
glycine
(Gly),
valine
(Val),
2-amino-
n
-butyric
acid
(
α
-ABA),
isovaline
(Iva)
and
norvaline
(Nva).
In the
present
study,
we
further
improved
our
3D-HPLC
system
for
the
determination
of eight
amino
acids
to reveal
their
ste
-
reochemistry
in
a variety
of
the
Ryugu
samples.
Two
proteinogenic
amino
acids,
aspartic
acid
(Asp)
and
serine
(Ser),
were
added
to the
previous
targets
in order
to compare
the
enantiomeric
ratios
between
proteinogenic
and
non-proteinogenic
amino
acids.
The
analytical
con
-
ditions
for
all
the
target
amino
acids
were
investigated,
and
the
appli
-
cability
of the
developed
system
was
examined
by
the
determination
of
amino
acid
enantiomers
in
four
different
carbonaceous
meteorites
(Murchison,
Aguas
Zarcas,
Tarda
and
Jbilet
Winselwan).
As
for
the
Ryugu
sample,
C0107
sample
was
used
in addition
to the
A0106
sample
(used
for
the
initial
analysis),
and
their
hot-water
extracts
and
hydro
-
chloric
acid
(HCl)
extracts
were
analyzed
to determine
the
enantiomeric
ratios
of the
amino
acids.
2. Materials
and methods
2.1.
Materials
Racemic
mixtures
of Ala
and
α
-ABA
were
obtained
from
FUJIFILM
Wako
Pure
Chemical
Corporation
(Osaka,
Japan).
DL
-Asp,
DL
‑
Ser
and
DL
-
Val
were
purchased
from
Nacalai
Tesque
(Kyoto,
Japan).
Gly
and
DL
-Nva
were
obtained
from
Sigma-Aldrich
(St.
Louis,
MO,
USA).
The
enantio
-
mers
of Iva
were
products
of AstaTech
(Bristol,
PA,
USA).
Acetonitrile
(MeCN)
of HPLC
grade
and
the
derivatization
reagent
for
fluorescence
detection,
4-fluoro-7-nitro-2,1,3-benzoxadiazole
(NBD-F),
were
ob
-
tained
from
Nacalai
Tesque
and
Tokyo
Chemical
Industry
(Tokyo,
Japan),
respectively.
Methanol
(MeOH)
of HPLC
grade,
boric
acid,
for
-
mic
acid
(FA),
trifluoroacetic
acid
(TFA)
and
sodium
hydroxide
(NaOH)
were
purchased
from
FUJIFILM
Wako
Pure
Chemical
Corporation.
Ul
-
trapure
water
and
ultrapure
hydrochloric
acid
(HCl)
for
sample
prepa
-
ration
were
products
from
Tama
Chemicals
(Kawasaki,
Japan).
Water
for
the
other
experiments
was
purified
using
a Milli-Q
Integral
3 system
(Merck,
Darmstadt,
Germany).
All
other
reagents
were
the
highest
grade
and
were
used
without
further
purification.
2.2.
Sample
extraction
Two
aggregate
Ryugu
samples,
A0106
(13.08
mg)
and
C0107
(10.73
mg),
consisting
of
<
1 mm
grains
were
used
in this
study.
The
A0106
sample
was
collected
from
the
asteroid
surface
during
the
first
touch
-
down.
The
C0107
sample
was
collected
during
the
second
touchdown
after
the
small
carry-on
impactor
experiment
[46], possibly
containing
subsurface
materials.
The
serpentine
powder
(16.21
mg),
which
was
prebaked
at 500
◦
C for
3 h, was
prepared
to check
a procedural
blank.
The
samples
were
extracted
with
hot
water
followed
by
aqueous
HCl
extraction
in the
clean
bench
(ISO
5) set
inside
the
cleanroom
(ISO
6)
[37]. All
of the
glassware
was
baked
at 500
◦
C for
3 h before
use
to
remove
any
possible
contamination
by
amino
acids.
The
hot-water
extraction
was
performed
by
adding
200
μ
L of water
to the
sample
powder
(A0106,
C0107
and
serpentine)
and
heated
at 105
◦
C for
20
h in
a nitrogen-purged
close
glass
ampoule.
After
the
extraction,
the
content
including
the
residue
and
solution
was
trans
-
ferred
to a glass
vial.
The
glass
vial
was
centrifuged
at 14,000
rpm
for
8
min,
and
the
supernatant
was
transferred
to
another
glass
vial.
The
ampoule
used
for
the
extraction
was
rinsed
with
water
(200
μ
L),
and
the
water
was
transferred
to
the
glass
vial
containing
the
residue.
After
shaking
and
centrifuging
at 14,000
rpm
for
8 min,
the
supernatant
was
transferred
to the
vial
containing
the
first
supernatant.
This
rinse
step
was
performed
twice,
and
the
obtained
supernatant
(total
600
μ
L) was
well
mixed
(hot-water
extract
solution).
To
250
μ
L of the
solution
(each
hot-water
extract
was
equivalent
to 5.45
mg
of A0106,
4.47
mg
of C0107
and
6.75
mg
of the
serpentine
blank),
250
μ
L of aqueous
HCl
(ca.
6 mol/
L) was
added
and
heated
at 105
◦
C for
20
h. The
reaction
mixture
was
evaporated
to dryness
under
reduced
pressure
and
used
for
the
amino
acid
analysis
by
3D-HPLC.
The
same
volumes
of the
hot-water
extracts
were
sent
for
a parallel
amino
acid
analysis
with
somewhat
different
hydrolysis
conditions
[38].
As
for
the
HCl
extraction,
half
of the
residue
obtained
after
the
hot-
water
extraction
was
mixed
with
200
μ
L of 3 mol/L
aqueous
HCl
in a
nitrogen-purged
glass
ampoule.
The
glass
ampoule
was
closed
and
heated
at 105
◦
C for
20
h, and
the
supernatant
was
transferred
to a glass
vial.
To
the
glass
ampoule
containing
the
residue,
200
μ
L of water
was
added,
and
the
glass
ampoule
was
ultrasonicated
for
15
min.
After
centrifugation,
the
supernatant
was
transferred
to the
same
glass
vial.
This
process
was
performed
twice,
and
a total
of 600
μ
L of the
super
-
natant
was
obtained
(HCl
extract
solution).
An
aliquot
(200
μ
L) of the
solution
was
evaporated
to dryness
and
utilized
for
the
3D-HPLC
anal
-
ysis
(each
HCl
extract
equivalent
to 2.18
mg
of A0106,
1.79
mg
of C0107
and
2.70
mg
of the
serpentine
blank).
Again,
the
same
volumes
of the
fractions
were
sent
for
a parallel
amino
acid
analysis
[38].
Four
meteorite
samples,
including
three
CM-type
(Murchison,
Aguas
Zarcas
and
Jbilet
Winselwan)
and
one
C-ungrouped
(Tarda)
carbona
-
ceous
chondrites,
were
used
in this
study.
While
the
Murchison
mete
-
orite
fell
in 1969,
the
Aguas
Zarcas
and
Tarda
meteorites
were
recent
falls
in 2019
and
2020,
respectively.
The
Jbilet
Winselwan
meteorite
was
found
from
the
Sahara
desert
in 2013.
The
inner
fragments
of the
meteorites
were
powdered
using
an
alumina
mortar
and
pestle.
The
meteorite
powders
were
extracted
with
hot
water
in the
same
manner
as
the
Ryugu
samples.
The
sample
amounts
were
22.0
mg
for
Murchison,
3.9
mg
for
Aguas
Zarcas,
3.7
mg
for
Jbilet
Winselwan
and
3.9
mg
for
Tarda.
2.3.
Derivatization
of the amino
acids
The
dried
Ryugu
extract
was
dissolved
in
100
μ
L of
water.
The
neutralization
of
the
sample
solution
was
performed
with
aqueous
NaOH
and
was
confirmed
by
a pH
test
paper.
Two
hundred
μ
L of water
was
added
to each
of the
Aguas
Zarcas,
Tarda
and
Jbilet
Winselwan
samples,
and
150
μ
L of the
solution
was
neutralized.
For
the
Murchison
meteorite,
after
200
μ
L of water
was
added,
50
μ
L of the
solution
was
diluted
five
times
by
water
and
neutralized.
An
aliquot
(20
μ
L) of the
neutralized
solution
was
mixed
with
400
mM
sodium
borate
buffer
(pH
8.0,
20
μ
L) and
100
mM
NBD-F
in MeCN
(5
μ
L) in a light-shielded
glass
vial.
The
mixed
solution
was
heated
at 60
◦
C for
6 min.
After
adding
an
aqueous
2 % (v/v)
TFA
solution
(55
μ
L),
10
μ
L of the
reaction
mixture
was
injected
into
the
3D-HPLC
system.
2.4.
3D-HPLC
conditions
The
HPLC
instruments
were
developed
using
the
NANOSPACE
SI-2
series
produced
by
Shiseido
(Tokyo,
Japan)
and
Singularity
series
designed
by
collaboration
with
KAGAMI
(Ibaraki,
Osaka,
Japan).
The
system
consisted
of the
NANOSPACE
3101
and
3201
pumps,
a 3202
degasser,
a 3033
auto-sampler,
3004
and
3014
column
ovens
and
a
Singularity
LED-3
fluorescence
detector
with
three
detection
cells.
NANOSPACE
high-pressure
valves
(3011
and
3012)
and
a 9986
multi-
loop
valve
(having
8 loops
of 300
μ
L) were
operated
by
a Singularity
A. Furusho
et al.
Journal of Chromatography Open 5 (2024) 100134
4
valve-controlling
system
to perform
the
multi-dimensional
analysis.
An
EZChrom
Elite
system
was
used
for
data
processing.
Fig.
1 represents
the
flow
diagram
of the
system.
A Singularity
RP18
column
(C18
bonded
silica
particle-packed
column,
3-
μ
m particle
size,
1.0
×
250
mm)
was
used
at 45
◦
C in the
first
dimension
for
the
reversed-phase
separation.
The
mobile
phase
A was
a mixed
solution
of TFA/MeCN/water
(0.025/
5/95,
v/v/v)
and
B was
a mixed
solution
of TFA/MeCN/water
(0.025/
25/75).
The
gradient
elution
was
carried
out
as follows:
100
% A for
45
min,
linear
gradient
elution
of 100
% A to 100
% B for
75
min
and
100
%
B for
30
min.
The
flow
rate
was
50
μ
L/min.
After
the
reversed-phase
separation,
the
target
amino
acids
were
separately
trapped
in
the
loops
of the
multi-loop
valve,
and
the
collected
fractions
were
sequen
-
tially
introduced
into
the
second
separation
column.
In the
second
dimension,
an
anion-exchange
column,
Singularity
AX
(aminopropylsilica
particle
packed
column,
5-
μ
m particle
size,
1.0
×
150
mm)
was
equipped
at 25
◦
C for
the
further
separation
of the
target
amino
acids
from
interferences.
The
mobile
phases
were
mixed
solutions
of
MeOH
and
MeCN
(50/50)
containing
FA
(0.04
% for
Ala,
α
-ABA,
Val
and
Nva,
0.07
% for
Ser,
Gly
and
Iva,
0.30
% for
Asp)
at the
flow
rate
of 100
μ
L/min.
The
target
amino
acids
were
again
fractionated
in the
loops
of the
multi-loop
valve
(having
two
loops
of 450
μ
L),
and
the
fractions
were
transferred
to an
enantioselective
column
representing
the
third
dimen
-
sion.
The
enantioselective
column
was
a tandemly
connected
Singularity
CSP-001S
column
(
N
-(3,5-dinitrophenylaminocarbonyl)-
L
-leucine
bonded
aminopropylsilica
particle
packed
column,
5-
μ
m particle
size,
1.5
×
250
mm,
total
length
was
500
mm)
maintained
at 25
◦
C. The
mobile
phases
were
mixed
solutions
of MeOH
and
MeCN
(50/50)
containing
FA
(0.15
% for
Ala,
α
-ABA,
Val
and
Nva,
0.30
% for
Ser
and
Gly,
0.70
% for
Asp).
For
the
enantiomer
separation
of Iva,
a mixed
solution
of MeOH
and
MeCN
(90/10)
containing
0.14
% FA
was
used
as the
mobile
phase.
The
flow
rate
was
200
μ
L/min.
The
NBD-amino
acids
were
quantified
by their
fluorescence
detection
(
λ
ex
=
470
nm,
λ
em
=
530
nm)
in all
dimensions.
3. Results
and discussion
3.1.
Development
and
evaluation
of an enantioselective
3D-HPLC
system
for extraterrestrial
amino
acids
Eight
α
-amino
acids
(
α
-ABA,
Ala,
Asp,
Gly,
Ser,
Val,
Iva
and
Nva)
were
selected
as the
target
molecules
for
the
Ryugu
samples
because
the
number
of analytes
for
the
3D-HPLC
system
was
limited.
Concerning
Asp
and
Ser,
they
are
abundantly
present
in the
terrestrial
environment,
and
the
large
excesses
of
their
L
-forms
are
well
known.
They
were
addi
-
tionally
chosen
in the
present
study
from
the
initial
analysis
to compare
the
chiral
property
between
proteinogenic
and
non-proteinogenic
amino
acids
and
evaluate
the
terrestrial
contamination.
All
the
selected
amino
acids
have
already
been
found
in several
carbonaceous
chondrites
[5,12,
14,32], though
some
studies
have
not
distinguished
the
enantiomers.
The
chromatograms
obtained
for
the
standard
amino
acids
with
the
present
3D-HPLC
system
are
shown
in Fig.
S1.
In the
first
dimension
(1D),
all
of the
target
amino
acids
were
separated
within
150
min
by
a
gradient
elution
of 5
–
25
% MeCN
0.025
% TFA
in H
2
O at the
flow
rate
of
50
μ
L/min.
The
target
amino
acids
were
individually
trapped
in the
multi-loops
at the
time
periods
indicated
by
the
black
bars
below
the
baseline
of the
1D
chromatogram
in Fig.
S1.
The
collected
fractions
were
sequentially
introduced
into
the
second
dimension
(2D).
The
fraction
volumes
were
162.5
μ
L (195
s) for
Ser,
200
μ
L (240
s) for
Asp,
150
μ
L
(180
s) for
Gly,
Iva,
Val
and
Nva,
125
μ
L (150
s) for
Ala
and
100
μ
L (120
s) for
α
-ABA.
The
target
amino
acids
in the
fractions
were
then
separated
from
interferences
by an anion-exchange
column.
The
FA
concentrations
in mobile
phases
(MeOH-MeCN,
90/10.
v/v)
were
investigated
in order
to obtain
the
appropriate
retention
times
of the
target
amino
acids.
The
selected
FA
concentrations
were
0.07
% for
Ser,
Gly
and
Iva,
0.30
% for
Asp,
0.04
% for
Ala,
α
-ABA,
Val
and
Nva.
With
these
mobile
phases,
all
the
amino
acids
eluted
within
20
min
and
were
fractionated
into
the
second
multi-loops
at the
time
periods
as indicated
by
black
bars
below
the
baseline
of 2D
chromatogram
in Fig.
S1.
The
fraction
volumes
were
300
μ
L (180
s) for
Ser,
275
μ
L (165
s) for
Asp,
200
μ
L (120
s) for
Gly
and
Nva,
225
μ
L (135
s) for
Ala,
α
-ABA,
Iva
and
Val.
The
collected
fractions
were
then
transferred
to
an
enantioselective
column
in
the
third
dimension
(3D).
Both
in the
first
and
second
dimensions,
relatively
large
volumes
of
the
fractions
(over
100
μ
L)
were
collected
and
trans
-
ferred/introduced
into
the
next
dimension.
However,
the
target
amino
acids
were
appropriately
retained
to the
columns,
and
their
peak
shapes
remained
sharp
enough
to be
fractionated
and
quantified.
These
results
indicated
that
the
mobile
phase
compatibility
issues
between
first-second
and
second-third
dimensions
were
acceptable
and
sufficient
for
the
quantitative
analyses.
Since
the
enantiomer
separation
of Iva
was
difficult,
two
Singularity
CSP-001S
columns
(1.5
×
250
mm)
were
tan
-
demly
connected
(total
length
was
500
mm)
as
described
above
to
obtain
a sufficient
separation.
After
the
thorough
mobile
phase
inves
-
tigation,
the
Iva
enantiomers
were
separated
within
60
min
by
using
Fig. 1.
Flow
diagram
of the
3D-HPLC
system.
C1,
Singularity
RP18
(1.0
×
250
mm);
C2,
Singularity
AX
(1.0
×
150
mm);
C3,
Singularity
CSP-001S
(1.5
×
500
mm).
AS,
auto
sampler;
CO,
column
oven;
D, detector;
HPV,
high
pressure
valve;
MLV,
multi-loop
valve;
P, pump;
W,
waste.
A. Furusho
et al.
Journal of Chromatography Open 5 (2024) 100134
5
0.14
%
FA
in
a mixed
solution
of
the
MeOH-MeCN
(90/10,
v/v).
Enantioselective
separations
of the
other
amino
acids
were
achieved
within
50
min
by
the
mobile
phases
of MeOH-MeCN
(50/50,
v/v)
so
-
lutions
containing
FA;
0.30
% for
Ser,
0.70
% for
Asp
and
0.15
% for
Ala,
α
-ABA,
Val
and
Nva.
The
obtained
resolution
factors
(
R
s) were
6.32
for
Ser,
1.77
for
Asp,
4.76
for
Ala,
4.10
for
α
-ABA,
1.13
for
Iva,
2.65
for
Val
and
4.15
for
Nva.
Although
the
Iva
enantiomers
were
not
completely
separated
(
R
s
<
1.5),
their
separation
was
practically
sufficient
to quan
-
tify
the
enantiomeric
ratio.
The
developed
3D-HPLC
system
was
validated
using
standard
solu
-
tions.
Because
the
contamination
of the
proteinogenic
L
-amino
acids
and
D
-Ala
from
reagents,
water
and
laboratory
wares
was
not
negligible,
the
calibration
curves
of these
amino
acid
enantiomers
were
constructed
in
the
range
from
5 to 100
fmol
to 10
pmol
depending
on
their
environ
-
mental
levels.
The
range
of Gly
was
from
200
fmol
to 20
pmol
because
its
concentration
in the
extraterrestrial
samples
is usually
higher
than
the
other
amino
acids.
For
the
other
amino
acid
enantiomers,
except
for
Iva,
the
calibration
curves
were
constructed
in the
range
from
1 fmol
to 10
pmol.
As
for
the
Iva
enantiomers,
the
calibration
range
was
set
from
4
fmol
to 40
pmol.
All
of the
calibration
curves
showed
good
linearities
with
a coefficient
of determination
(
r
2
) higher
than
0.9989
(Table
1).
The
lowest
points
of the
calibration
curves
were
adopted
as the
lower
limit
of quantification
(LLOQ;
1 fmol
for
D
-Ser,
D
-Asp,
D
,
L
-
α
-ABA,
D
-Val
and
D
,
L
-Nva,
4 fmol
for
D
,
L
-Iva,
5 fmol
for
D
-Ala
and
L
-Val,
10
fmol
for
L
-
Ser,
L
-Asp
and
L
-Ala,
and
200
fmol
for
Gly).
For
the
precision,
the
results
of five
continuous
analyses
were
evaluated
as the
between-run
precision
using
low
and
high
concentrations
of the
standard
amino
acid
solutions.
Since
the
total
analysis
time
for
the
full
3D-HPLC
determination
was
more
than
six
hours,
the
intra-day
precision
could
not
be
checked.
The
obtained
relative
standard
deviation
(RSD)
values
of the
between-run
precision
were
2.15
–
6.56
% for
the
low
concentration
and
0.33
–
8.38
% for
the
high
concentration
of the
standard
amino
acids.
The
system
validation
was
also
made
with
the
Murchison
carbonaceous
chondrite.
The
analytical
results
of the
hot-water
extract
of the
Murchison
mete
-
orite
are
summarized
in Table
1. The
amino
acids
in the
Murchison
meteorite
were
determined,
and
the
RSD
values
were
4.37
–
7.40
% (
n
=
5).
As
for
the
recovery
of the
amino
acids,
two
concentrations
of the
standard
amino
acids
were
added
to the
hot-water
extract
of Murchison,
and
the
obtained
accuracy
values
for
the
low
and
high
concentrations
were
89.4
–
104.6
% and
89.0
–
107.8
%,
respectively.
Several
multi-dimensional
GC
or LC
methods
have
been
developed
by
combining
more
than
two
separation
modes
to
obtain
the
higher
selectivity
for
enantiomeric
separation
of the
amino
acids
[39,44,47].
The
2D-GC-MS
method
consisted
of a polyethylene
glycol-based
column
and
an
enantioselective
column,
and
19
chiral
amino
acids
in
the
Murchison
meteorite
sample
were
separated
and
determined
[47].
Concerning
multi-dimensional
LC,
even
though
several
2D-
and
3D-HPLC
methods
have
been
developed
for
the
determination
of trace
amounts
of amino
acids
in the
Murchison
and
Antarctic
carbonaceous
meteorites
[39,44], the
present
3D-HPLC
system
was
improved
for
the
enantioselective
analysis
of
trace
levels
of
the
extraterrestrial
amino
acids.
The
current
3D-HPLC
system
enables
the
separation
of the
Ala,
α
-ABA,
Iva,
Val
and
Nva
enantiomers
(with
R
s values
of
1.13
–
4.76)
equal
to or greater
than
the
previous
study
(
R
s values
from
1.04
to 4.65)
[44], and
Ser
and
Asp
enantiomers
were
also
separated
with
sufficient
R
s
values
(6.32
for
Ser
and
1.77
for
Asp).
The
ranges
of the
calibration
curves
were
expanded
from
those
of the
previous
methods,
especially
for
the
non-proteinogenic
amino
acid
enantiomers
(0.004
–
40
pmol
for
Iva,
0.001
–
10
pmol
for
α
-ABA
and
Nva),
and
the
validation
of the
developed
method
was
successfully
performed
with
good
linearities
of the
cali
-
bration
curves,
practically
low
RSD
values
of the
precision
and
a suffi
-
cient
accuracy.
These
results
showed
that
the
present
3D-HPLC
system
is
highly-selective,
sensitive
and
quantitative
for
the
determination
of
chiral
amino
acids
in the
extraterrestrial
samples.
The
current
3D-HPLC
system
can
precisely
quantify
the
8 target
amino
acids,
while
the
OPA/NAC
method
(also
used
for
the
analysis
of
Ryugu
sample)
can
detect
most
amino
acids
using
one
separation
col
-
umn
[38]. Although
the
non-targeted
amino
acid
analysis
was
per
-
formed
in a relatively
short
analytical
time
using
the
OPA/NAC
method,
the
most
effective
use
of the
3D-HPLC
system
is to precisely
quantify
each
enantiomer
of
the
amino
acids
due
to
its
high
selectivity.
The
three-dimensional
separation
results
in
a significant
decrease
of
the
unknown
interfering
peaks
for
the
detection
of trace
levels
of the
amino
acids.
3.2.
Chiral
amino
acids
in carbonaceous
meteorites
The
validated
3D-HPLC
system
was
applied
to the
determination
of
amino
acids
in the
carbonaceous
meteorites
(Murchison,
Aguas
Zarcas,
Tarda
and
Jbilet
Winselwan)
as rehearsal
analyses
of the
Ryugu
sample.
Table
1
Method
validation
for
the
enantioselective
determination
of 8 target
amino
acids.
Amino
acids
Standard
Murchison
meteorite
Calibration
curves
Precision
(RSD,%)
Precision
(RSD,%)
Accuracy
(%)
Calibration
range
(pmol)
Equation
r
2
Low
High
Low
High
D
-Ser
0.001
–
10
y
=
19.8
x
+
0.06
0.9999
2.29
1.56
4.92
95.9
91.9
L
-Ser
0.01
–
10
y
=
12.9
x
+
0.19
0.9999
2.24
1.55
5.03
89.4
89.0
D
-Asp
0.001
–
10
y
=
15.5
x - 0.46
0.9993
2.38
1.29
7.40
94.2
95.3
L
-Asp
0.01
–
10
y
=
12.2
x - 0.64
0.9989
2.15
1.10
7.36
93.6
94.6
Gly
0.2
–
20
y
=
25.7
x
+
1.29
0.9998
2.85
1.02
5.71
94.0
91.5
D
-Ala
0.005
–
10
y
=
20.8
x - 0.52
0.9997
2.97
0.33
6.33
94.3
97.0
L
-Ala
0.01
–
10
y
=
15.4
x - 0.60
0.9995
2.36
0.45
6.38
94.2
96.6
D
-
α
-ABA
0.001
–
10
y
=
24.7
x - 0.60
0.9997
3.09
1.06
4.99
94.9
96.7
L
-
α
-ABA
0.001
–
10
y
=
19.2
x - 0.48
0.9997
3.02
0.90
5.10
95.0
97.2
D
-Iva
0.004
–
40
y
=
2.9
x
+
0.55
0.9992
4.90
8.38
6.38
103.9
95.8
L
-Iva
0.004
–
40
y
=
2.8
x
+
0.54
0.9993
5.03
8.31
6.49
104.6
96.1
D
-Val
0.001
–
10
y
=
25.0
x - 0.32
1.0000
2.32
2.11
4.39
97.5
100.0
L
-Val
0.005
–
10
y
=
21.2
x - 0.33
1.0000
2.34
1.97
4.37
97.8
99.7
D
-Nva
0.001
–
10
y
=
23.8
x - 0.65
0.9997
6.56
3.53
6.98
95.5
107.6
L
-Nva
0.001
–
10
y
=
18.4
x - 0.56
0.9996
6.41
3.49
6.84
92.1
107.8
Equations
were
made
where
x was
the
injection
amount
of the
amino
acids
(pmol)
and
y was
the
peak
height
(mV).
Injection
amounts
of
D
-Ser,
D
-Asp,
D
,
L
-
α
-ABA,
D
-Val
and
D
,
L
-Nva
were
0.001,
0.005,
0.01,
0.1,
1, 5 and
10
pmol.
Those
of
L
-Ser,
L
-Asp
and
L
-Ala
were
0.01,
0.1,
1, 5 and
10
pmol.
Those
of Gly
were
0.2,
2, 10
and
20
pmol.
Those
of
D
-Ala
and
L
-Val
were
0.005,
0.01,
0.1,
1, 5 and
10
pmol.
Those
of
D
- and
L
-Iva
were
0.004,
0.02,
0.04,
0.4,
4, 20
and
40 pmol.
For
the
precision
of the
standard
(
n
=
5),
0.2
and
5 pmol
of racemic
amino
acids
and
Gly
were
injected
(0.8
and
20
pmol
were
used
for
DL
-Iva).
For
the
precision
of the
carbonaceous
meteorite
(
n
=
5),
the
intrinsic
amino
acids
in the
Murchison
meteorite
were
determined.
For
the
accuracy,
1 or 2.5
pmol
of
DL
-Ser,
DL
-Asp,
DL
-Val
and
DL
-Nva,
2 or 5 pmol
of Gly
and
DL
-Ala,
0.4
or 1 pmol
of
DL
-
α
-ABA
and
0.1
or 0.25
pmol
of
DL
-Iva
were
added
to the
Murchison
meteorite.
A. Furusho
et al.