Simultaneous determination of five N-alkylamides in the root of Anacyclus pyrethrum by HPLC and profiling of components in its methanolic root extract by UPLC/Q-TOF-MS

Abstract

The root of Anacyclus pyrethrum (L.) Lag., Asteraceae, is very widely used for treating various diseases in Traditional Uygur Medicine, particularly in the treatment of vitiligo. However, there have been few studies on the quality standards of A. pyrethrum in China. A. pyrethrum contains abundant N-alkylamides, which are considered to be the principal components. Therefore, based on the previous research in our group, six N-alkylamides were obtained by using column chromatography. We used ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry to determine the mass spectrometry cleavage mechanism of these six monomer components and established the mass spectrometry cleavage law of N-alkylamides. Then, we used the ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry method to rapidly identify and analyze the N-alkylamide components of the A. pyrethrum methanol extract. Finally, twenty N-alkylamides were identified, including eleven N-isobutylamides, two N-methyl isobutylamides, six 4-hydroxyphenylethyl-amide and one 2-phenylethylamide. Five of these compounds were identified as new compounds that have not been reported to date. Two of these compounds were identified for the first time in this herb. Therefore, this work provides an approach for the quality analysis of N-alkylamides in the root of A. pyrethrum. A search of the literature showed that the content determination in the A. pyrethrum quality standard is still a remaining problem. N-alkylamides are the main components of A. pyrethrum. Even though ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry has the advantages of lower time and higher efficiency compared to high-performance liquid chromatography, considering the ease of repeatability and universality of the quality control method, we chose to use high-performance liquid chromatography for content determination. In this experiment, high-performance liquid chromatography was used for the first time to establish a simple, rapid and accurate method for evaluating the N-alkylamide content in A. pyrethrum with five N-alkylamides used as the standards. Finally, this work provides a qualitative and quantitative method for the analysis of N-alkylamides in A. pyrethrum, improving the quality control standards for A. pyrethrum.

Full text

Revista
Brasileira
de
Farmacognosia
29
(2019)
152–161
ww
w
.
elsevier.com/locate/bjp
Original
Article
Simultaneous
determination
of
five
N-alkylamides
in
the
root
of
Anacyclus
pyrethrum
by
HPLC
and
profiling
of
components
in
its
methanolic
root
extract
by
UPLC/Q-TOF-MS
Ruifang
Ji
,
Qinghua
Quan
,
Xiaoyu
Guo
,
Jiamei
Zhang
,
Yongli
Song
,
Mengting
Zhu
,
Peng
Tan
,
Jing
Han ∗,
Yonggang
Liu ∗
Beijing
University
of
Chinese
Medicine,
Beijing
100102,
China
a
r
t
i
c
l
e
i
n
f
o
Article
history:
Received
12
March
2018
Accepted
7
December
2018
Available
online
20
March
2019
Keywords:
N-alkylamides
Quantitative
analysis
Qualitative
analysis
a
b
s
t
r
a
c
t
The
root
of
Anacyclus
pyrethrum
(L.)
Lag.,
Asteraceae,
is
very
widely
used
for
treating
various
diseases
in
Traditional
Uygur
Medicine,
particularly
in
the
treatment
of
vitiligo.
However,
there
have
been
few
studies
on
the
quality
standards
of
A.
pyrethrum
in
China.
A.
pyrethrum
contains
abundant
N-alkylamides,
which
are
considered
to
be
the
principal
components.
Therefore,
based
on
the
previous
research
in
our
group,
six
N-alkylamides
were
obtained
by
using
column
chromatography.
We
used
ultra-performance
liquid
chromatography
quadrupole
time-of-flight
mass
spectrometry
to
determine
the
mass
spectrom-
etry
cleavage
mechanism
of
these
six
monomer
components
and
established
the
mass
spectrometry
cleavage
law
of
N-alkylamides.
Then,
we
used
the
ultra-performance
liquid
chromatography
quadrupole
time-of-flight
mass
spectrometry
method
to
rapidly
identify
and
analyze
the
N-alkylamide
compo-
nents
of
the
A.
pyrethrum
methanol
extract.
Finally,
twenty
N-alkylamides
were
identified,
including
eleven
N-isobutylamides,
two
N-methyl
isobutylamides,
six
4-hydroxyphenylethyl-amide
and
one
2-
phenylethylamide.
Five
of
these
compounds
were
identified
as
new
compounds
that
have
not
been
reported
to
date.
Two
of
these
compounds
were
identified
for
the
first
time
in
this
herb.
Therefore,
this
work
provides
an
approach
for
the
quality
analysis
of
N-alkylamides
in
the
root
of
A.
pyrethrum.
A
search
of
the
literature
showed
that
the
content
determination
in
the
A.
pyrethrum
quality
standard
is
still
a
remaining
problem.
N-alkylamides
are
the
main
components
of
A.
pyrethrum.
Even
though
ultra-
performance
liquid
chromatography
quadrupole
time-of-flight
mass
spectrometry
has
the
advantages
of
lower
time
and
higher
efficiency
compared
to
high-performance
liquid
chromatography,
considering
the
ease
of
repeatability
and
universality
of
the
quality
control
method,
we
chose
to
use
high-performance
liquid
chromatography
for
content
determination.
In
this
experiment,
high-performance
liquid
chro-
matography
was
used
for
the
first
time
to
establish
a
simple,
rapid
and
accurate
method
for
evaluating
the
N-alkylamide
content
in
A.
pyrethrum
with
five
N-alkylamides
used
as
the
standards.
Finally,
this
work
provides
a
qualitative
and
quantitative
method
for
the
analysis
of
N-alkylamides
in
A.
pyrethrum,
improving
the
quality
control
standards
for
A.
pyrethrum.
©
2019
Sociedade
Brasileira
de
Farmacognosia.
Published
by
Elsevier
Editora
Ltda.
This
is
an
open
access
article
under
the
CC
BY-NC-ND
license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Introduction
Anacyclus
pyrethrum
(L.)
Lag.
belongs
to
the
Asteraceae
fam-
ily,
and
it
is
a
perennial,
procumbent
herb
that
is
covered
with
long
villous
and
reaches
15–45
cm
in
length,
and
is
widely
dis-
tributed
in
North
Africa,
Central
Asia
and
the
Xinjiang
region
of
China
(Pharmacopoeia
Commission
of
the
Ministry
of
Health,
1999;
Annalakshmi
et
al.,
2012;
Kumar
and
Lalitha,
2012).
In
research
∗Corresponding
authors.
E-mails:
hanjing8585@163.com
(J.
Han),
liuyg0228@163.com
(Y.
Liu).
carried
out
at
The
University
of
Macau,
ZhiQiao
et
al.
(2014)
found
that
this
Uyghur
medicine
not
only
was
very
widely
used
in
Xin-
jiang
but
also
had
a
profound
impact
in
South
Asia,
Central
Asia
and
Afghanistan.
It
is
mainly
used
for
the
treatment
of
diseases
such
as
tumors,
knee
osteoarthritis
(Chinese
Materia
Medica
Committee,
2005),
and
particularly
for
treatment
of
vitiligo
(Mubarak
and
Aiguuli,
2004;
Genaguri
and
Mairemu,
2011).
Due
to
its
remarkable
role,
it
has
received
increasing
attention,
but
its
use
faces
prob-
lems
such
as
insufficient
research
regarding
pharmacodynamics
and
quality
control.
According
to
the
literature
(Yongmin,
1999),
among
these,
N-alkylamides
are
considered
to
be
the
principal
components
of
A.
pyrethrum,
and
current
research
has
focused
on
https://doi.org/10.1016/j.bjp.2018.12.011
0102-695X/©
2019
Sociedade
Brasileira
de
Farmacognosia.
Published
by
Elsevier
Editora
Ltda.
This
is
an
open
access
article
under
the
CC
BY-NC-ND
license
(http://
creativecommons.org/licenses/by-nc-nd/4.0/).

R.
Ji
et
al.
/
Revista
Brasileira
de
Farmacognosia
29
(2019)
152–161
153
the
separation
and
analysis
of
N-alkylamides.
Boonen
et
al.
(2011)
isolated
four
N-alkylamides
from
A.
pyrethrum
while
Boonen
et
al.
(2012b)
identified
thirteen
N-alkylamides
in
the
ethanol
extract
of
A.
pyrethrum.
N-alkylamide
is
an
amide
structure
composed
of
an
amine
with
a
longer
unsaturated
acid
at
one
end
and
an
amine
with
a
smaller
substituent
at
the
other
end,
and
it
is
an
important
natural
product
widely
present
in
plants
such
as
Asteraceae
and
Aristolochia
species.
N-alkylamides
are
considered
to
be
a
promising
group
of
bioactive
compounds
that
possess
multiple
pharmacolog-
ical
activities,
such
as
antimicrobial
activities,
tingling
and
related
organoleptic
effects,
anti-inflammatory
and
immunomodulatory
effects,
and
tyrosinase
activation
effect
(Badhe
et
al.,
2010;
Boonen
et
al.,
2012a;
Sharma
et
al.,
2013).
The
research
on
quality
control
is
highly
significant
for
ensur-
ing
the
safety
of
traditional
Chinese
medicine.
Traditional
Chinese
medicine
does
not
exert
its
effects
through
the
action
of
one
or
even
several
components.
Rather,
its
effectiveness
is
the
result
of
the
interaction
of
various
components.
As
the
commonly
used
medicine
for
the
treatment
of
vitiligo
in
Uygur
medicine,
A.
pyrethrums
have
been
rarely
studied
for
quality
standards,
par-
ticularly
regarding
the
selection
and
content
determination
of
the
indicator
components.
Currently,
although
some
of
the
N-alkylamides
in
A.
pyrethrum
have
been
separated
and
analyzed,
these
results
are
still
insuffi-
cient.
Hence,
further
research
on
the
composition
of
A.
pyrethrum
is
highly
necessary.
Generally,
the
traditional
techniques
of
extrac-
tion
and
separation
of
compounds
are
suitable.
However,
it
is
difficult
to
isolate
the
minor
and
trace
constituents
by
using
the
traditional
techniques.
The
previously
reported
methods
for
analyzing
N-alkylamides
in
medicinal
plants
are
based
on
High-
Performance
Liquid
Chromatography
coupled
with
Mass
Spectra
(HPLC-MS)
(Boonen
et
al.,
2010,
2012b;
Sharma
et
al.,
2013).
While
HPLC-MS
has
some
advantages
in
medicinal
herb
component
anal-
ysis,
the
sample
separation
process
in
HPLC
is
time-consuming
and
the
low-resolution
MS
instrument
does
not
allow
the
exact
measurement
of
the
masses
of
the
precursor
and
fragment
ions
to
obtain
the
highest
reliability
in
structural
identifica-
tion.
In
recent
years,
UPLC-Q-TOF-MS
has
been
shown
to
be
a
rapid
and
convenient
method,
and
it
has
started
to
play
a
signifi-
cant
role
in
the
identification
of
complex
components
system,
compensating
for
the
shortcomings
of
the
HPLC-MS
method.
The
Q-TOF-MS
has
high
MS/MS
sensitivity,
enabling
accurate
mass
measurement
of
the
precursor
and
fragment
ions,
and
it
reaches
the
lowest
minimum
detection
limit
of
any
other
high
resolution
LC/MS
method.
Therefore,
UPLC/Q-TOF-MS
has
become
a
powerful
tool
for
rapid
separation,
screening
and
identification
of
complex
systems
of
traditional
medicine
sam-
ples.
In
our
previous
work,
we
isolated
the
dichloromethane
frac-
tion
of
A.
pyrethrum
with
silica
gel
column
chromatography
and
obtained
six
N-alkylamides.
The
cleavage
mechanism
of
the
N-
alkylamide
monomer
compounds
isolated
from
A.
pyrethrum
was
analyzed
and
the
regularity
of
mass
spectrometry
was
estab-
lished.
UPLC/Q-TOF-MS
was
used
to
rapidly
identify
and
analyze
the
N-alkylamides
in
the
methanol
extract
of
A.
pyrethrum.
Iden-
tification
of
N-alkylamides
was
completed
based
on
the
known
fragmentation
patterns
and
literature
data.
Finally,
the
five
isolated
N-alkylamide
were
selected
as
index
components
and
quantita-
tively
analyzed
by
HPLC.
We
sought
to
achieve
quantitative
and
qualitative
analysis
of
N-alkylamides
from
the
root
of
A.
pyrethrum.
Our
study
can
provide
a
basis
for
improving
the
quality
standards
of
A.
pyrethrum.
Materials
and
methods
Materials
The
dried
fruits
of
Anaycclus
pyerhturm
(L.)
Lag.,
Asteraceae,
were
purchased
from
the
Haozhou
Medicinal
Corporation,
Anhui,
China,
with
a
batch
number
of
130704.
Voucher
specimens
were
identified
by
professor
Muaitaer
of
Collece
of
Xinjiang
Uyghur
Medicine.
Acetonitrile
and
methanol
were
obtained
from
Fisher
Sci-
entific
(USA)
and
were
of
HPLC
grade.
The
water
used
was
Watsons
distilled
water,
and
all
other
reagents
were
of
analytical
grade.
Stan-
dards
of
deca-2E,4E-dienoic
acid
4-hydroxyphenylethylamide
(1),
deca-2E,4E-dienoic
acid
isobutyl-amide
(2),
dodeca-2E,4E-dienoic
acid
4-hydroxyphenylethylamide
(3),
tetradeca-2E,4E,8E-trienoic
acid
4-hydroxyphenylethylamide
(4),
tetradeca-2E,4E-dienoic
acid
4-hydroxyphenylethylamide
(5),
and
undeca-2E,4E-diene-8,10-
diynoic
acid
2-phenylethyl
amide
(6)
were
extracted
and
separated
in
our
laboratory.
The
purity
of
each
compound
was
not
less
than
98%,
as
specified
by
HPLC
analysis.
Apparatus
and
method
Standard
sample
preparation
through
column
chromatography
The
air-dried
powder
of
A.
pyrethrum
(2
kg)
was
extracted
three
times
(each
for
1.5
h)
with
70%
ethanol.
The
filtrate
was
concentrated
under
vacuum
to
obtain
an
ethanol
extract
(272
g).
The
ethanol
extract
was
dissolved
in
water
and
extracted
with
petroleum
ether
and
dichloromethane
in
turn.
The
dichloromethane
extract
(40
g)
was
obtained
and
then
separated
by
silica
gel
column
chromatography
with
a
gra-
dient
elution
of
petroleum
ether-ethyl
acetate
(10:1–1:1).
After
the
further
separation
and
purification
using
C18 and
Sephadex
LH-20
column
chromatography,
compounds
1–6
were
obtained.
Using
ultraviolet,
nuclear
magnetic,
and
mass
spec-
trometry,
we
identified
these
six
components:
deca-2E,4E-dienoic
acid
4-hydroxyphenylethylamide
(1,
22
mg),
deca-2E,4E-dienoic
acid
isobutyl-amide
(2,
20
mg),
dodeca-2E,4E-dienoic
acid
4-
hydroxyphenylethylamide
(3,
10
mg),
tetradeca-2E,4E,8E-trienoic
acid
4-hydroxyphenylethylamide
(4,
5
mg),
tetradeca-2E,4E-
dienoic
acid
4-hydroxyphenylethyl
amide
Tetradeca-2E,4E-dienoic
acid
4-hydroxyphenylethyl
amide
(5,
5
mg)
and
undeca-2E,4E-
diene-8,10-diynoic
acid
2-phenylethyl
amide
(6,
10
mg).
The
six
standard
samples
were
stored
for
one
month
in
the
dark
at
a
tem-
perature
below
4◦C.
Among
these
six
compounds,
compound
4
was
a
novel
compound
and
compounds
5
and
6
were
isolated
for
the
first
time
from
this
plant.
The
NMR
spectrum
of
compound
4
is
provided
in
the
supplementary
material.
Qualitative
analysis
of
components
by
UPLC-Q-TOF-MS
Sample
preparation.
Methanol
(approximately
25
ml)
was
added
to
A.
pyrethrum
(0.5
g),
and
the
mixture
was
ultrasonicated
(Kun-
shan
Ultrasonic
Instrument
Co.,
Ltd.,
Kunshan,
China,
100
kHz)
and
extracted
for
1
h.
The
sample
solution
was
filtered
through
a
0.45
␮m
filter
membrane.
The
filtered
solution
was
diluted
10-fold.
UPLC
conditions.
UPLC
analysis
was
performed
using
an
ACQUITY
UPLCTM HSS
C18 column
(100
mm
×
2.1
mm,
1.8
␮m,
Waters
Co.,
Milford,
MA,
USA).
The
mobile
phase
consisted
of
0.1%
acid
water
(A)
and
acetonitrile
(B)
(v/v)
with
gradient
elution
separation
as
described
in
Table
1.
Q-TOF-MS
conditions.
The
ACQUITY
UPLC
system
was
coupled
to
a
hybrid
quadrupole
orthogonal
time-of-flight
(Q-TOF)
mass

154
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Brasileira
de
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29
(2019)
152–161
Table
1
Mobile
phase
composition
in
UPLC.
Time
(min)
A%
(0.1%
acid
water
%)
B%
(acetonitrile
%)
0
40
60
1
40
60
10
5
95
12
5
95
12.1
40
60
Table
2
Mobile
phase
composition
in
HPLC.
Time
(min)
A%
(water
%)
B%
(methanol
%)
0
30
70
12
30
70
14
20
80
40
20
80
spectrometer
(Synapttm
G2
HDMS,
waters,
Manchester,
U.K.)
equipped
with
electrospray
ionization
(ESI),
and
leucine-
enkephalin
with
accurate
mass
was
used
as
the
correction
fluid.
The
operating
parameters
were
as
follows:
capillary
voltage
3.0
kV
(ESI+)
or
2.2
kV
(ESI−);
sample
cone
voltage
40
V;
extraction
cone
voltage
4
V,
source
temperature
100 ◦C,
desolvation
temperature
400 ◦C
and
desolvation
gas
flow
800
l/h.
In
the
MSE
mode,
the
trap
collision
energy
of
the
low
energy
function
was
set
to
6
eV,
while
the
ramp
trap
collision
energy
of
the
high
energy
function
was
set
at
10–40
eV.
To
ensure
mass
accuracy
and
reproducibility,
the
mass
spectrometer
was
calibrated
over
the
range
of
100–1500
Da
with
sodium
formate.
Leucine-enkephalin
(m/z
556.2771
in
the
positive
ion
mode;
m/z
554.2615
in
the
negative
ion
mode)
was
used
as
the
external
reference
of
Lock-SprayTM infused
at
a
constant
flow
of
5
␮l/min.
The
data
acquisition
mode
was
3D
data
acquisition
under
the
Continuum
mode.
A
V4.1
Mass
Lynx
data
processing
station
was
used
for
data
analysis.
Quantitative
analysis
of
N-alkylamides
by
HPLC
Sample
preparation.
Standards
stock
solutions
were
prepared
by
accurately
dissolving
each
component
in
5
ml
of
methanol
and
storing
at
4◦C.
Mixed
standard
solutions
of
the
five
N-alkylamides
were
obtained
at
concentration
of
10,
29.3,
8.8,
0.86,
and
3.4
g/ml,
respectively.
This
solution
was
used
for
method
development
and
validation.
Anaycclus
pyrethrum
(approximately
0.5
g)
was
extracted
by
ultrasound
(Kunshan
Ultrasonic
Instrument
Co.,
Ltd.,
Kunshan,
China,
100
kHz)
with
methanol
(25
ml)
for
1
h.
The
sample
solu-
tion
was
filtered
through
a
0.45
␮m
filter
membrane.
The
filtered
sample
solution
(10
␮l)
was
directly
injected
into
the
HPLC
system.
Deca-2E, 4E-dienoicacid 4-hydroxyphenylethylamide
Compound 1 ([M+H]+=288) Compound 2 ([M+H]+=224)
Compound 4 ([M+H]+=342)
Compound 3 ([M+H]+=316)
Compound 5 ([M+H]+=344) Compound 6 ([M+H]+=278)
Deca-2E, 4E-dienoicacid isobutylamide
Tetradeca-2E, 4E, 8
E-trienoic acid 4-hydroxyphenylethylamide
Tetradeca-2E, 4E-dienoic acid 4-hydroxyphenylethylamide Undeca,2E,4
E-diene-8,10-diynoic acid 2-phenylethylamide
Dodeca-2E, 4
E-dienoicacid 4-hydroxyphenylethylamide
100
100
100
0
0
0
0
120 120
120
120
100
100
100
140 140
140
140
160 160
160
160
180 180
180
180
200
200
200
m/z
m/z
m/z m/z
m/z
m/z
121.0638
121.0638
121.0638
121.0638 128.0610
133.1051 168.1398
196.1696
207.1747
146.0970 173.1073189.1638
100
161.1370
122.0700 121.1023
129.0710
157.0671 168.0831
165.0009 177 1636
187.1514
205.1622
120
120
140
140
160
160
180
180
200
200
100
100
0
0
179.1451
169.1434
151.1161
168.1398
123.1191
123
151.1161
%
%
%
%
%%
A
B
C
E
F
D
Fig.
1.
MS2fragmentation
spectra
of
the
six
N-alkylamides
separated
in
Anacyclus
pyrethrum.
(A)
Deca-2E,4E-dienoic
acid
4-hydroxyphenylethylamide
(1);
(B)
Deca-2E,4E-
dienoic
acid
isobutyl-amide
(2);
(C)
Dodeca-2E,4E-dienoic
acid
4-hydroxyphenylethylamide
(3);
(D)
Tetradeca-2E,4E,8E-trienoic
acid
4-hydroxyphenylethylamide
(4);
(E)
Tetradeca-2E,4E-dienoic
acid
4-hydroxyphenylethyl
amide
(5);
and
(F)
Undeca-2E,4E-diene-8,10-diynoic
acid
2-phenylethyl
amide
(6).

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155
Table
3
Compounds
1–13
identified
in
Anacyclus
pyrethrum
extract
by
UPLC/Q-TOF-MS.
Compound
Rt
(min)
Precursor
ion
(m/z)
Cal.
(M+H)
Productions
(m/z)
Structural
formula
Identification
Structure
7
2.07
230.1589
230.1545
174,
157,
131,
129,
116
C15H19 NO
Undeca-2E,4E-diene-8,10-
diynoic
acid
IBA
6
2.41
278.1564
278.1545
157,
141,
131,
129,
103
C16H29 NO
Undeca-2E,4E-diene-8,10-
diynoic
acid
2-phenylethyl
amide
8
2.51
244.1706
244.1701
180,
174,
157,
166,
131,
129,
114,
103
C16H21 NO
Undeca-2E,4E-diene-8,10-
diynoic
acid
N-Me
IBA
1
2.59
288.2002
288.1964
168,
151,
133,
123,
121,
119,
105
C18H25 NO2Deca-2E,4E-dienoic
acid
4-
hydroxyphenylethylamide
9
3.76
224.2031
224.2014
168,
151,
133,
123,
119,
C14H25 NO
Deca-2E,4E-dienoic
acid
IBA
(pellitorine)
3
4.17
316.2322
316.2277
196,
179,
161,
133,
119
C20H29 NO2Dodeca-2E,4E-dienoic
acid
4-
hydroxyphenylethylamide
10
4.39
272.2027
272.2014
216,
173,
171,
131,
C18H25 NO
Tetradeca-2E,4E-diene-
8,10-diynoic
acid
IBA
(anacycline)
11
4.58
238.2027
238.2171
182,
168,
151,
133,
123,
119,
109
C15H27 NO
Deca-2E,4E-dienoic
acid
N-Me
IBA
4
4.82
342.2451
324.2433
205,
187,
177,
173,
145,
131,
121,
C22H31 NO2Tetradeca-2E,4E,8E-
trienoic
acid
4-
hydroxyphenylethylamide
12
5.3
276.2373
276.2327
220,
203,
173,
152,
135,
C18H29 NO
Tetradeca-2E,4E,
XE/Z,
YE/Z-tetraenoic
IBA

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Table
3
(Continued)
Compound
Rt
(min) Precursor
ion
(m/z) Cal.
(M+H) Productions
(m/z)
Structural
formula
Identification
Structure
2
5.79 252.2324 252.2327 196,
161,
179,
119 C16H29 NO
Dodeca-2E,4E-dienoic
acid
isobutyl-amide
5
6.18 344.2594 344.259 224,
207,
189,
172,
147,
133,
121,
119
C22H33 NO2Tetradeca-2E,4E-dienoic
acid
4-hydroxyphenylethyl
amide
13
8.05
280.265
280.264
224,
207,
182,
154,
147,
133,
141
C18H33 NO
Tetradeca-2E,4E-dienoic
acid
IBA
HPLC
conditions.
HPLC
analysis
was
performed
using
a
Shimadzu
LC-20A
system
(Shimadzu,
Corporation,
Kyoto,
Japan),
config-
ured
with
a
PDA
detector,
a
quaternary
gradient
pump
and
an
autosampler.
The
data
analysis
was
performed
using
the
Shimadzu
“LC
Lab-Solution”
software
(Shimadzu
Corporation,
Kyoto,
Japan).
Separation
was
performed
using
an
Agilent
Extend
C18 column
(4.6
mm
×
250
mm,
5
␮m)
at
a
flow
rate
of
0.8
ml
min−1.
The
mobile
phase
consisted
of
water
(A)
and
methanol
(B)
(v/v).
A
gradient
program
was
adopted
as
described
in
Table
2.
Accuracy,
reproducibility,
stability
and
recovery
rate
tests.
The
accu-
racy
test
included
intra-day
and
inter-day
analyses
that
were
used
to
evaluate
the
method
precision.
Mixed
standards
solution
(10
␮l)
were
precisely
drawn,
injected
continuously
six
times
over
three
days,
and
their
peak
areas
were
measured.
Stability
tests
were
further
performed
to
analyze
the
variations
in
the
sample
solutions
over
10
h.
Sample
solutions
(10
␮l)
were
precisely
drawn,
injected
continuously
at
0,
2,
4,
6,
8,
and
10
h,
and
their
peak
areas
were
measured.
Reproducibility
tests
were
further
performed
to
test
the
repeatability
of
the
method.
Six
test
solutions
(10
␮l)
were
con-
tinuously
injected
into
a
high-performance
liquid
chromatography
instrument,
and
their
peak
areas
were
measured.
Recovery
tests
were
also
performed
to
validate
the
accuracy
of
the
developed
method
by
adding
a
known
amount
of
the
reference
marker
compounds
into
accurately
weighed
samples.
The
spiked
samples
were
extracted
using
the
sample-preparation
method,
and
then
the
developed
HPLC
method
was
used
for
analysis.
Results
and
discussion
Rapid
analysis
of
methanolic
root
extract
by
UPLC/Q-TOF-MS
Establishment
of
MS
fragmentation
pathways
of
N-alkylamides
It
was
reported
that
there
are
four
main
types
of
N-alkylamides
in
A.
pyrethrum,
namely,
isobutylamide
(IBA),
N-methyl
isobuty-
lamide
(N-methyl
isobutylamide),
2-phenylethylamide
(2-PEA)
and
4-hydroxyphenylethylamide
(4-OH
PEA).
However,
there
have
been
few
reports
on
the
fragmentation
pathways
of
N-alkylamides,
and
the
corresponding
pathways
are
often
presented
by
only
one
reference
standard
(Boonen
et
al.,
2011;
Sharma
et
al.,
2013).
There-
fore,
to
obtain
accurate
and
comprehensive
information
about
the
fragmentation
pathways
of
the
N-alkylamides,
compounds
1–6
were
analyzed
by
Q-TOF-MS.
The
characteristic
fragment
ions
were
identified
and
the
patterns
were
deduced.
In
the
positive
ionization
mode,
the
[M+H]+peak
is
prone
to
␣-cracking,
both
in
the
N-position
and
the
carbonyl-position,
and
different
types
of
N-alkylamides
produce
different
characteristic
fragment
ions.
Furthermore,
most
N-alkylamides
contain
poly-
unsaturated
aliphatic
fatty
acid
chain
that
produces
one
or
more
fragmentation
ions
with
the
loss
of
m/z
14
(CH2).
The
IBA
presents
the
characteristic
fragment
losses
of
56,
73,
and
101
(Fig.
1B).
The
2-PEA
presents
the
characteristic
fragment
losses
of
121,
133,
and
151
(Fig.
1F).
The
4-OH
PEA
has
the
characteristic
fragmentation
losses
of
120,
137,
155,
and
165
(Fig.
1B–E).
The
MS2fragmentation
spectra
of
the
six
N-alkylamides
separated
from
A.
pyrethrum
were
elucidated
(Fig.
1,
Table
3).
Rapid
analysis
of
the
compounds
in
Anacyclus
pyrethrum
The
identification
of
N-alkylamides
in
the
A.
pyrethrum
extract
required
two
steps.
First,
a
preliminary
identification
was
per-
formed
by
combining
the
precise
molecular
weight
of
each
compound
that
was
obtained
by
high-resolution
mass
spectrome-
try;
second,
based
on
the
determination
of
the
MS
fragmentation
pathways
of
the
N-alkylamides,
a
further
structural
inference
was

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157
Table
4
Compounds
14–20
detected
in
Anacyclus
pyrethrum
extract
by
UPLC/Q-TOF-MS.
Compound
Rt
(min)
Precursor
ion
(m/z)
Cal.
(M+H)
Productions
(m/z)
Structural
formula
Identification
Structure
14
1.59
294.1532
294.1494
246,
163,
157,
129,
121
C19H19 NO2Undeca-2E,4E-diene-8,10-
diynoic
acid
4-hydroxy-
phenylethylamide
15
2.81
258.1892
258.1858
202,
172,
157,
131,
117,
105
C17H23 NO
(2E,7Z)-N-isobutyl-2,7-
tridecadiene-10,12-
diynamide
16
2.93
222.1874
222.1858
172,
166,
149,
123,
121,
C14H23 NO
(2E,6Z,8E)-N-isobutyl-
2,6,8-decatrienamide
17
3.16
314.2166
314.212
190,
177,
151,
149
C20H27 NO2Dodeca-2E,4E,
nE-
trienoic
acid
4-
hydroxyphenylethylamide
18
3.48
270.1894
270.1858
214,
197,
171,
169,
129,
115,
128,
113
C18H23 NO
Tetradeca-2E,4E,
nE-trienoic-8,10-diynoic
acid
IBA
19
4.29
274.2163
274.2171
201,
173,
159
C18H27 NO
Tetradeca-2E-diny-8,10-
diynoic
acid
IBA
20
6.55
278.2511
278.2484
222,
205,
167,
152,
141
C18H31 NO
Tetradeca-2E,
4E,
nE-trienoic
acid
IBA

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100
%
0
2.00
4.00
6.00
8.00
10.00
12.00
Time
13
11
12
10
9
8
6
5
F7E
CDB
4
23
1
A
G
Fig.
2.
Base
peak
intensity
of
Anacyclus
pyrethrum
in
the
positive
ionization
mode.
Undeca-2E, 4E-diene-8,10-diynoicacid 4-OHphenylethylamide
Compound 17 ([M+H]+=294) Compound 18 ([M+H]+=258)
Compound 19([M+H]+=222)
Compound 20([M+H]+=314)
Compound 21([M+H]+=270)
Compound 22([M+H]+=274)
Tetradeca-2E,4
E, n
E-trienoic -8,10-diyonoic acid IBA
Tetradeca-2E-diny-8,10-diynoic acid IBA
Tetradeca -2E, 4E, nE-trienoic acid IBA Compound 23 ([M+H]+=278)
Deca-2E,4
E-dienoicacid 4-hydroxyphenylethylamide
Deca-2E,4
E-dienoicacid 4-hydroxyphenylethylamide
(2E,7
Z)-N-isobuty1-2,7-tridecadiene -10,12-diynamide
100
0
120 130
130
140
140
150
100 110 120
160 170 180 190 200 210
150 160 170 180 190 200 210
230220210200190180170160150140130120
220
222.1874
205.1622
222
172.8661
166.1246
167.1335
167
124.0802
152.1107
107.0892
113.9648
119.0878
128.0659 141.0744
152.1107 172.8661
153.0708
154.0772 173.8722
202.8734 228
214
197
179.0691
173.8722
165.1300
177 121
149.1367
144.8699
128.8726
121.0686
172.8661
166.1302
162.8821
157.8725
149.0994
121
144.8699
H3C
166
202.8734
173.8722
142.0823
157.1054
115.0581
113.9648
172.8661
202
157
121
00
133.0900
131.0878
128.8776
121.1023
121.0686
128.0659
121.0686
129.0710 144.8699 157.0671
129
228.7963
144.8699 159.1203
170.1557
172.8661
173.8722
179.1451
201.0429
203.0476
m/z
m/z
m/z
m/z
m/z
m/z
m/z
%
100
100
100
100
100 100
0
0
0
0
140 150
150145140135125120
110 120 160120130 140 140150 160 180 200170
155 160 165 170
160 170 180 190120 130
130
%%%%%
%
A
C
D
E
F
G
B
Fig.
3.
MS2fragmentation
spectra
of
compounds
14–20
identified
in
Anacyclus
pyrethrum
extract.

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159
100
0
2.00
4.00
H
6.00
8.00
10.00
Time
%
Fig.
4.
Base
peak
intensity
of
Anacyclus
pyrethrum
in
the
negative
ionization
mode.
Di-acyl caffeoylquinic acids Compound24([M+H]-=515)
191.0650
179.0434
100
100
0
%
150 200 250
350 400
m/z
354.0945
315.0573
192.0688
353.0923
300
Fig.
5.
MS2fragmentation
spectra
of
compound
21.
made.
In
the
positive
ionization
mode,
20
N-alkylamides
were
iden-
tified
in
the
A.
pyrethrum
extract:
eleven
IBA,
two
N-Me
IBA,
six
4-OH
PEA
and
one
2-PEA.
The
base
peak
intensity
chromatogram
of
the
main
components
is
presented
in
Figs.
1,
2,
and
Table
4
illus-
trate
the
seven
novel
N-alkylamides
identified
in
the
A.
pyrethrum
extract.
UPLC/Q-TOF-MS
detected
twenty
N-alkylamides
in
the
A.
pyrethrum
extract.
Compounds
1–13
have
been
reported
in
the
literature
and
were
unambiguously
identified
based
on
their
frag-
mentation
patterns
and
literature
data.
Table
3
presents
their
retention
time,
precursor
ion
and
fragmentation
information,
as
well
as
their
structural
information.
Another
seven
compounds
found
in
A.
pyrethrum
that
have
not
been
reported
previously
were
obtained.
Table
4
and
Fig.
2
present
their
retention
times
(Rt),
pre-
cursor
ions,
calculated
mass,
produced
ions,
and
proposed
structures.
Compound
14
displayed
ion
peaks
at
m/z
157,
m/z
129
and
m/z
103,
which
are
the
characteristics
of
an
undeca-
2E,4E-diene-8,10-diynoic
acid
moiety,
respectively.
Compounds
15
(m/z
258)
and
16
(m/z
222),
two
known
N-alkylamides,
were
first
discovered
in
A.
pyrethrum
and
identified
as
(2E,7Z)-
N-isobutyl-2,7-tridecadiene-10,12-diynamide
and
(2E,6Z,8E)-N-
isobutyl-2,6,8-decatrien-amide,
respectively.
Compound
17
was
identified
as
a
4-OH
PEA
(m/z
314)
and
had
typical
ion
peaks
at
m/z
177
and
m/z
149,
consistent
with
dodec-dienoic
acid
4-
hydroxyphenylethyl-amide.
Compounds
18
and
19
were
identified
as
IBA
based
on
their
fragmentation
patterns
that
correspond
to
anacycline
(m/z
272).
The
structural
formulae
of
compounds
18
and
19
with
m/z
270
and
274
are
C18H23 NO
and
C18H27 NO,
respec-
tively.
The
4-OH
PEA
possesses
typical
fragmentation
losses
of
−120,
−137
and
−165.
Compound
20
showed
the
characteristic
fatty
acid
fragmentation
signal
at
m/z
205
that
was
consistent
with
the
tetradeca-trienoic
acid
moiety.
The
structural
formulae
of
17
and
20
were
C20H27 NO2and
C18H31 NO,
respectively,
but
the
loca-
tion
of
the
third
saturated
bond
could
not
be
assigned
under
the
LC–MS
conditions
(Fig.
3).
In
the
negative
ionization
mode,
one
compound
was
identi-
fied.
The
BPI
of
A.
pyrethrum
is
presented
in
Fig.
4.
Compound
21
was
identified
as
di-acyl
caffeoylquinic
acid.
The
MS2fragmenta-
tion
spectrum
of
compound
H
is
shown
in
Fig.
5.
The
spectrum
of
compound
H
showed
the
precursor
ion
of
m/z
515,
and
some
mass
fragments
at
m/z
353,
191,
179,
and
135
that
had
a
good
match
with
di-acyl
caffeoylquinic
acids,
but
the
specific
structure
of
the
compound
could
not
be
confirmed
under
the
LC–MS
conditions.
mAU
mAU
241nm,4nm (1.00)
241nm,4nm (1.00)
75
50
25
25
0
0
0.0
0.0
5.0
1
1
2
2
3
3
4
4
5
5
50
10.0
15.0
20.0
25.0
30.0
35.0
t/mIn
5.0
10.0
15.0
20.0
25.0
30.0
35.0
t/mIn
A
B
Fig.
6.
HPLC
chromatograms
of
the
methanol
extract
of
the
root
of
Anacyclus
pyrethrum
(A)
and
a
mixed
standard
solution
of
the
five
N-alkylamides
(B).
Deca-2E,4E-dienoic
acid
4-hydroxyphenylethylamide
(1);
Deca-2E,4E-dienoic
acid
isobutylamide
(2);
3.
Dodeca-2E,4E-dienoic
acid
4-hydroxyphenylethylamide
(3);
4.
Tetradeca
-2E,4E,8E-trienoic
acid
4-hydroxyphenylethylamide
(4);
5.
Tetradeca-2E,
4E-dienoic
acid
4-hydroxyphenylethyl-amide
(5).

160
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29
(2019)
152–161
mAU
80
70
60
50
40
30
20
10
0
200
250 300
350 nm
5
4
3
1
2
240
242442
z44
40
Fig.
7.
Absorption
spectra
of
the
five
N-alkylamides:
Deca-2E,4E-dienoicacid
4-hydroxyphenylethylamide
(1);
Deca-2E,
4E-dienoic
acid
isobutylamide
(2);
Dodeca-2E,
4E-dienoicacid
4-hydroxyphenylethylamide
(3);
Tetradeca-2E,4E,8E-
trienoic
acid
4-hydroxyphenylethylamide
(4);
Tetradeca-2E,4E-dienoic
acid
4-
hydroxyphenylethyl
amide
(5).
Simultaneous
determination
of
five
N-alkylamides
in
the
root
of
A.
pyrethrum
by
HPLC
Optimization
of
quantitative
analysis
of
five
N-alkylamides
To
achieve
the
maximum
extraction
efficiency
of
the
five
N-
alkylamides,
three
parameters
including
extraction
methods,
the
amount
of
extraction
solvent
and
extraction
time
were
compared
individually.
The
results
showed
that
for
the
extraction
methods,
ultrasonic
extraction
had
a
higher
efficiency
than
reflux
extraction.
Using
methanol
(25
ml)
and
1
h
extraction
allowed
the
full
extrac-
tion
for
analysis.
Therefore,
we
adopted
ultrasonic
extraction
for
1
h
by
methanol
(25
ml)
for
the
analysis.
HPLC
conditions
were
optimized
by
changing
the
HPLC
gradient
program.
The
selected
program
described
in
the
HPLC
conditions
section
above
was
shown
to
have
high
separation
performance
on
C18 column.
Fig.
6
shows
the
typical
HPLC
chromatogram
of
the
root
of
A.
pyrethrum.
Methodological
inspection
Calibration
curves,
limit
of
detection
(LOD),
and
limit
of
quantifi-
cation
(LOQ).
Six
different
concentrations
of
standard
solutions
were
injected
to
determine
the
calibration
curve.
Quantification
of
the
test
compounds
was
performed
using
the
external
stan-
dard
method.
The
detector
response
was
set
at
241
nm,
where
each
compound
presented
its
maximum
absorption
(Fig.
7).
The
correlation
coefficients
(r)
of
all
standard
components
(within
the
range
of
0.9997–0.999)
showed
good
linearity.
By
comparing
the
signal-to-noise
ratios
of
threefold
(3␴)
and
tenfold
(10␴)
varia-
tions,
the
limit
of
detection
(LOD)
and
limit
of
quantification
(LOQ)
of
the
five
analytes
were
calculated.
The
LOD
and
LOQ
ranges
were
0.44–3.73
and
1.19–12.44
ng,
respectively.
The
obtained
results
are
described
in
detail
in
Table
5.
Accuracy,
reproducibility,
stability
and
recovery
rate
tests.
The
RSD
was
used
as
a
measure
of
the
accuracy,
repeatability,
stability
and
recovery
rate.
The
RSD
value
of
the
accuracy
tests
was
less
than
1.67%.
Therefore,
it
was
determined
that
the
instrument
has
good
precision
and
meets
the
experimental
requirements.
The
RSD
value
of
the
stability
tests
was
less
than
1.43%.
Therefore,
it
was
deter-
mined
that
the
sample
solution
is
stable
within
10
h.
The
RSD
value
of
the
reproducibility
tests
was
smaller
than
1.81%.
Therefore,
it
was
determined
that
the
method
is
reproducible
and
meets
the
experi-
mental
requirements.
The
RSD
value
of
the
recovery
tests
was
less
than
2.79%.
The
results
of
the
four
experiments
are
listed
in
Table
6,
confirming
that
this
method
was
appropriate
for
analysis.
Determination
of
sample
content
Anaycclus
pyrethrum
samples
were
prepared
in
triplicate
and
were
injected
into
the
HPLC
system
for
content
determination.
The
results
are
shown
in
Table
7.
Conclusion
UPLC/Q-TOF-MS
was
applied
for
qualitative
profiling
of
the
components
of
A.
pyrethrum
root
extract.
N-alkylamides
present
explicitly
characteristic
fragmentation
pathways
that
can
be
used
as
a
basis
for
the
identification
of
the
compo-
nents
in
A.
pyrethrum.
Using
this
approach,
21
compounds
were
identified,
including
twenty
N-alkylamides
and
one
organic
Table
5
Regression
data,
limit
of
detection
(LOD),
and
limit
of
quantification
(LOQ)
of
the
five
analytes
by
HPLC.
Analyte
Regression
equation
r
Linear
range
(␮g)
LOD
(ng)
LOQ
(ng)
1
y
=
83176x
−
4896.8
0.9999
10.00–400.00
0.44
1.46
2
y
=
205274x
+
143461
0.9999
29.33–1173.20
3.73
12.44
3
y
=
62833x
−
2853.5
0.9997
8.80–352.00
0.37
1.24
4
y
=
9436.1x
−
1749
0.9998
0.86–34.40
0.35
1.19
5
y
=
16982x
−
4020.1
0.9997
3.40–136.00
1.40
4.68
Table
6
Precision,
repeatability,
stability
and
recovery
rate
of
the
five
analytes
by
HPLC.
Precision
Repeatability
Stability
Recovery
rate
Analyte
(RSD,
%)
(RSD,
%,
n
=
6)
(RSD,
%,
n
=
6)
(n
=
6)
Intraday
(n
=
6)
Interday
(n
=
3)
Mean
RSD
(%)
1
0.21
0.63
0.90
0.71
99.69%
1.57
2
0.55
0.69
0.64
0.59
96.23%
2.79
3
0.43
0.46
1.59
1.06
96.04%
2.28
4
1.67
1.28
1.81
1.43
101.12%
2.03
5
0.65
0.59
1.72
0.81
105.06%
2.46

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Farmacognosia
29
(2019)
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161
Table
7
Contents
of
five
N-alkylamide
based
on
HPLC
method
in
the
root
of
Anacyclus
pyrethrum
samples
(n
=
3).
Analyte
1
2
3
1
0.040%
0.042%
0.040%
2
0.128%
0.132%
0.131%
3
0.049%
0.050%
0.049%
4
0.003%
0.003%
0.003%
5
0.018%
0.019%
0.018%
acid.
(2E,7Z)-N-isobutyl-2,7-tridecadiene-10,12-diynamide
and
(2E,6Z,8E)-N-isobutyl-2,6,8-decatrienamide
were
reported
for
the
first
time
in
this
plant.
Undeca-2E,4E-diene-8,10-diynoic
acid
4-OH-phenylethylamide,
dodeca-2E,4E,E-trienoic
acid
4-hydroxyphenylethylamide,
tetradeca-2E,4E,nE-trienoic-
8,10-diynoic
acid,
tetradeca-2E-diny-8,10-diynoic
acid
and
tetradeca-2E,4E,nE-trienoic
acid
were
identified
as
new
com-
pounds
under
the
UPLC-Q-TOF-MS
conditions.
Based
on
the
literature
and
the
analysis
of
the
mass
spectrometry
results,
it
was
determined
that
the
N-alkylamides
are
the
main
chemical
components
in
A.
pyrethrum.
Therefore,
five
N-alkylamides
were
selected
as
the
index
components
for
HPLC
determination.
In
this
study,
a
simple
and
accurate
HPLC
method
for
the
simultaneous
separation
and
quantitative
analysis
of
five
N-alkylamides
in
the
root
of
A.
pyrethrum
was
established
for
the
first
time.
This
method
has
excellent
linearity,
precision,
stability,
and
accuracy,
and
can
be
employed
for
the
quality
evaluation
of
A.
pyrethrum.
In
conclusion,
our
study
should
be
valuable
for
qualitative
and
quantitative
analysis
of
A.
pyrethrum
and
can
improve
the
quality
control
standards.
Authorship
RJ,
determination
of
sample
solution
and
wrote
the
article;
QQ
and
XG,
separation
of
compounds;
JZ
and
YS,
extraction
of
sam-
ple
solution;
MZ,
literature
search;
PT,
instructor
and
guidance
of
the
experiments;
JH,
instructor
and
corrections
in
the
article;
YL,
instructor
and
design
of
the
experimental
plan.
Conflicts
of
interest
The
authors
declare
no
conflicts
of
interest.
Acknowledgments
This
work
was
supported
by
Natural
Science
Foundation
of
Xin-
jiang
(No.
201318101-5).
Appendix
A.
Supplementary
data
Supplementary
data
associated
with
this
article
can
be
found,
in
the
online
version,
at
doi:10.1016/j.bjp.2018.12.011.
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