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at
Journal
of
Pharmaceutical
and
Biomedical
Analysis
j
o
u r
n a
l
h o
m e p a g e :
w w w . e l s e v i e r . c o m / l o c a t e / j p b a
Short
communication
Simultaneous
determination
of
rutin
and
ascorbic
acid
in
a
sequential
injection
lab-at-valve
system
Mohammed
khair
E.A.
Al-Shwaiyat
,
Yuliia
V.
Miekh
,
Tatyana
A.
Denisenko
,
Andriy
B.
Vishnikin
,
Vasil
Andruch
,
Yaroslav
R.
Bazel
a
Department
of
Basic
and
Applied
Science,
Zarka
University
College,
Al-Balqa
Applied
University,
Al-Salt,
19117,
Jordan
b
Department
of
Analytical
Chemistry,
Faculty
of
Chemistry,
Oles
Honchar
Dnipro
National
University,
49010,
Dnipro,
Ukraine
c
Department
of
Analytical
Chemistry,
Faculty
of
Science,
P.J. ˇSafárik
University,
SK-01454
Koˇsice,
Slovak
Republic
a
r
t
i
c
l
e
i
n
f
o
Article
history:
Received
5
July
2017
Received
in
revised
form
25
October
2017
Accepted
1
November
2017
Available
online
6
November
2017
Keywords:
Sequential
injection
lab-at-valve
Simultaneous
determination
18-Molybdo-2-phosphate
Folin-Ciocalteu
reagent
Rutin
Ascorbic
acid
a
b
s
t
r
a
c
t
A
green,
simple,
accurate
and
highly
sensitive
sequential
injection
lab-at-valve
procedure
has
been
devel-
oped
for
the
simultaneous
determination
of
ascorbic
acid
(Asc)
and
rutin
using
18-molybdo-2-phosphate
Wells-Dawson
heteropoly
anion
(18-MPA).
The
method
is
based
on
the
dependence
of
the
reaction
rate
between
18-MPA
and
reducing
agents
on
the
solution
pH.
Only
Asc
is
capable
of
interacting
with
18-MPA
at
pH
4.7,
while
at
pH
7.4
the
reaction
with
both
Asc
and
rutin
proceeds
simultaneously.
In
order
to
improve
the
precision
and
sensitivity
of
the
analysis,
to
minimize
reagent
consumption
and
to
remove
the
Schlieren
effect,
the
manifold
for
the
sequential
injection
analysis
was
supplemented
with
external
reaction
chamber,
and
the
reaction
mixture
was
segmented.
By
the
reduction
of
18-MPA
with
reducing
agents
one-
and
two-electron
heteropoly
blues
are
formed.
The
fraction
of
one-electron
heteropoly
blue
increases
at
low
concentrations
of
the
reducer.
Measurement
of
the
absorbance
at
a
wavelength
corre-
sponding
to
the
isobestic
point
allows
strictly
linear
calibration
graphs
to
be
obtained.
The
calibration
curves
were
linear
in
the
concentration
ranges
of
0.3–24
mg
L
−1
and
0.2–14
mg
L
−1
with
detection
limits
of
0.13
mg
L
−1
and
0.09
mg
L
−1
for
rutin
and
Asc,
respectively.
The
determination
of
rutin
was
possible
in
the
presence
of
up
to
a
20-fold
molar
excess
of
Asc.
The
method
was
applied
to
the
determination
of
Asc
and
rutin
in
ascorutin
tablets
with
acceptable
accuracy
and
precision
(1–2%).
©
2017
Elsevier
B.V.
All
rights
reserved.
1.
Introduction
By
introducing
a
reaction
chamber
(RC)
into
the
flow
manifold,
the
obtained
system
exploits
the
characteristics
of
both
flow
and
batch
systems.
As
a
result,
such
a
system
combines
the
advan-
tages
of
the
automated
control
of
flows,
including
high
sampling
frequency,
complete
and
precise
control
of
reactant
volumes
and
timing
of
operations,
low
cost,
low
consumption
of
the
reagents
and
low
effluent
production
−
thus,
the
principles
considered
of
green
analytical
chemistry
−
with
the
wide
application
range
typical
for
batch
systems
Although
systems
in
which
RC
is
incorporated
into
sequential
injection
analysis
(SIA)
manifold
were
attributed
to
flow-batch
analysis
(FBA)
systems
is
more
logical
to
describe
them
as
∗ Corresponding
author
at:
Department
of
Analytical
Chemistry,
Faculty
of
Chem-
istry,
Oles
Honchar
Dnipro
National
University,
49010,
Dnipro,
Ukraine.
address:
(A.B.
Vishnikin).
a
separate
technique.
In
accordance
with
this,
the
“Lab-at-valve”
(LAV)
concept
was
introduced
by
Grudpan
the
SI-LAV
system,
sample
processing,
chemical
reaction
and/or
detection
are
carried
out
in
a
designed
LAV
unit
attached
to
the
port
of
a
multiposition
selection
valve.
A
LAV
unit
can
be
easily
fabricated
with
relatively
low-cost
materials
and
available
instrument/machine
tools.
As
follows
from
new
trends
in
existing
flow
methods,
increasing
attention
is
being
paid
to
multi-component
analysis.
Nevertheless,
a
review
of
the
literature
shows
that
contrary
to
the
numerous
developments
in
flow
injection
analysis
(FIA)
a
limited
number
of
papers
have
appeared
in
this
field
dealing
with
other
techniques,
including
SIA
FBA
Rutin
is
a
haemostatic
drug
used
in
the
treatment
of
diseases
characterised
by
capillary
bleeding
and
increased
capillary
fragility
is
often
used
together
with
ascorbic
acid
(Asc),
and
in
this
combination,
it
reduces
capillary
permeability
and
fragility
more
efficiently,
also
due
to
the
inhibition
of
hyaluronidase
activity.
Rutin
belongs
to
the
group
of
bioflavonoids,
and
in
line
with
Asc
it
participates
in
redox
processes.
Both
compounds
have
antiox-
https://doi.org/10.1016/j.jpba.2017.11.006
0731-7085/©
2017
Elsevier
B.V.
All
rights
reserved.
180
M.k.E.A.
Al-Shwaiyat
et
al.
/
Journal
of
Pharmaceutical
and
Biomedical
Analysis
149
(2018)
179–184
idant
properties
and
co-exist
in
plants.
A
few
methods
for
the
simultaneous
determination
of
Asc
and
rutin
in
their
combined
dosages
have
been
reported,
including
chemometric-assisted
UV
spectrophotometry
methods
the
SIA
technique
coupled
with
solid
phase
extraction
Recently,
the
ammonium
salt
of
18-molybdo-2-phosphate
het-
eropoly
anion
P
2
Mo
18
O
62
6
−
(18-MPA)
was
proposed
as
a
reagent
for
the
determination
of
reducing
agents,
and
several
simple,
fast,
automated,
sensitive
and
rather
selective
sequential
injec-
tion
methods
have
been
developed
for
the
determination
of
some
reducing
compounds
such
as
Asc,
p-aminophenol,
epinephrine
and
cysteine
In
the
present
work,
a
novel,
simple,
highly
sensitive,
envi-
ronmentally
friendly,
and
cost-effective
SI-LAV
method
has
been
developed
for
the
simultaneous
determination
of
Asc
and
rutin.
The
chemistry
used
in
the
determination
is
based
on
the
dependence
of
the
reaction
rate
between
18-MPA
as
reagent
and
reducing
agents
on
solution
pH.
Only
Asc
is
capable
of
interacting
with
18-MPA
at
pH
4.7,
while
at
pH
7.4
the
reaction
with
both
Asc
and
rutin
proceeds
simultaneously.
2.
Experimental
2.1.
Reagents
and
apparatus
Ultrapure
water
was
produced
by
a
Millipore
TM
water
purifi-
cation
system
(Millipore,
Bedford,
MA,
USA)
and
was
then
used
throughout
the
experiments.
The
ammonium
salt
of
18-MPA
(NH
4
)
6
P
2
Mo
18
O
62
×
14H
2
O
(18-MPC)
was
synthesized
and
recrys-
tallized
as
previously
reported
0.01
M
solution
of
18-MPC
was
prepared
by
dissolving
0.7855
g
of
the
salt
in
water
and
dilut-
ing
to
25
mL.
L-ascorbic
acid
(>99.7%
purity),
rutin
trihydrate
(>99%
purity),
methanol
(for
HPLC,
>
99.9%),
disodium
hydrogen
phos-
phate
dodecahydrate,
and
sodium
dihydrogen
phosphate
dihydrate
were
purchased
from
Fluka
Analytical
(Sigma-Aldrich,
Buchs,
Switzerland).
A
sample
of
the
0.01
M
ascorbic
acid
stock
solu-
tion
was
prepared
by
dissolving
an
accurately
weighed
amount
in
methanol.
The
stock
solution
of
1
mM
rutin
was
prepared
by
dissolving
and
diluting
66.4
mg
of
C
27
H
30
O
16
×
3H
2
O
to
a
final
vol-
ume
of
100
mL
with
methanol.
Both
of
the
last-mentioned
solutions
were
preserved
in
a
refrigerator
to
prevent
untimely
oxidation
with
oxygen
dissolved
in
solvent.
The
Asc
and
rutin
solutions
were
thus
stable
for
at
least
two
or
four
days,
respectively.
In
order
to
prevent
the
untimely
oxidation
of
rutin
and
ascorbic
acid
during
the
anal-
ysis,
the
dissolved
oxygen
was
removed
from
the
water
used
for
the
preparation
of
the
standard
and
sample
solutions
by
purging
with
nitrogen
at
a
flow
rate
of
25
mL
s
−1
for
30
min.
The
follow-
ing
commercially
available
Ascorutin
®
tablets
were
analysed:
1)
100
mg
of
Asc
and
20
mg
of
rutin
trihydrate
per
0.5
g
tablet
(Zen-
tiva,
Prague,
Czech
republic)
and
2)
50
mg
of
Asc
and
50
mg
of
rutin
trihydrate
per
0.33
g
tablet
(Kyiv
vitamin
factory,
Kyiv,
Ukraine).
An
acetate
buffer
solution
with
pH
4.7
±
0.2
was
prepared
by
mix-
ing
10.1
g
of
sodium
acetate
and
4.0
mL
of
glacial
acetic
acid
in
a
250
mL
flask
and
filling
up
to
the
mark
with
water.
The
phosphate
buffer
solution
of
pH
7.4
±
0.1
was
prepared
by
dissolving
1.17
g
of
NaH
2
PO
4
×
2H
2
O
and
7.78
g
of
Na
2
HPO
4
×
12H
2
O
in
water
and
filling
up
to
a
volume
of
500
mL
(final
concentrations
0.03
M
and
0.087
M
in
NaH
2
PO
4
and
Na
2
HPO
4
,
respectively).
The
absorbance
measurements
were
performed
on
a
Lightwave
II
UV–vis
spec-
trophotometer
(Biochrom
Ltd.,
Cambridge,
UK)
with
a
1.0
cm
quartz
cell.
An
Orion
720A
pH
meter
(Orion
Research
Co.,
Boston,
MA,
USA)
was
used
for
measuring
the
pH.
2.2.
SI-LAV
system
A
commercial
FIAlab
®
3500
system
(FIAlab
®
Instruments
Inc.,
Bellevue,
WA,
USA)
equipped
with
a
syringe
pump
(syringe
reservoir
5
mL)
and
an
8-port
selection
Cheminert
valve
(Valco
Instrument
Co.,
Houston,
TX,
USA)
was
used.
This
SIA
set-up
was
supplemented
with
an
LS–1
tungsten
halogen
lamp
as
the
visi-
ble
light
source,
a
USB4000-UV-VIS
diode
array
spectrophotometer
(both
Ocean
Optics
Inc.,
Dunedin,
FL,
USA),
and
a
microvolume
SMA-Z
flow
cell
with
a
20
mm
optical
path
length.
The
entire
SIA
system
was
controlled
by
the
FIAlab
software
package
(version
5.0).
Flow
lines
were
made
from
0.75
mm
i.d.
PTFE
tubing.
A
2
mL
microcentrifuge
polypropropylene
tube
with
1.2
cm
i.d.
width
and
a
funnel-shaped
inlet
at
the
bottom
was
used
as
the
reaction
cham-
ber.
The
SIA
manifold
used
for
the
simultaneous
determination
of
Asc
and
rutin
is
shown
schematically
in
2.3.
General
SI-LAV
procedure
The
overall
analytical
procedure
consisted
of
four
stages:
wash-
ing
the
RC,
delivering
the
reaction
components
into
the
RC,
carrying
out
the
chemical
reaction
and
measuring
the
analytical
signal.
At
the
first
stage,
the
flow-rate
is
set
at
100
L
s
−1
;
the
syringe
pump
valve
is
switched
to
position
IN;
and
the
syringe
pump
is
filled
with
1500
L
of
ultra
pure
water
used
as
the
carrier
solution.
Next,
the
syringe
pump
valve
is
switched
to
position
OUT,
and
450
L
of
water
is
driven
into
the
RC
through
port
2
of
the
multi-position
valve.
By
the
reverse
movement
of
the
syringe
pump
(500
L)
the
washing
is
first
directed
back
into
holding
coil
(HC)
and
then
into
the
waste
reservoir
through
port
1
(600
L).
At
the
second
stage,
the
flow-rate
is
reduced
to
50
L
s
−1
,
and
150
L
of
air
is
aspirated
into
the
HC
through
port
8,
followed
by
250
L
of
sample
and
40
L
of
0.15
mM
18-MPC
introduced
through
ports
5
and
port
6,
respectively.
After
that,
20
L
of
buffer
solution
with
pH
4.7
±
0.2
(port
3)
or
pH
7.4
±
0.2
(port
4)
are
drawn
into
the
HC.
The
obtained
mixture
is
moved
into
the
RC
with
360
L
of
water,
thus
leaving
100
L
of
air
in
HC.
Isolation
of
the
reaction
mixture
from
the
carrier
is
necessary
to
retain
sample
homogeniza-
tion.
At
the
third
stage,
560
L
of
air
is
introduced
into
the
HC,
and
then
570
L
of
air
is
passed
through
the
solution
in
the
RC.
In
this
way,
the
fully
homogenized
solution
is
obtained
at
the
ear-
liest
possible
time.
In
order
to
complete
the
reduction
of
18-MPA
with
analytes,
the
reaction
mixture
is
maintained
for
240
s.
At
the
measurement
stage,
the
spectrometer
reference
scan
is
made.
The
coloured
solution
is
first
dispensed
into
the
HC
(400
L),
and
then
320
L
of
this
solution
is
forced
out
through
port
7
into
the
Z-flow
cell
at
30
L
s
−1
(at
higher
flow
rates
the
probability
of
the
appearance
of
bubbles
on
the
walls
of
flow
cell
increases),
and
the
flow
is
stopped
for
20
s.
The
measured
absorbances
are
averaged
during
this
time
period.
The
response
is
measured
at
920
nm.
Finally,
the
remaining
solution
and
the
water
contained
in
the
system
are
directed
through
port
1
to
the
waste
reservoir
by
emptying
the
syringe
pump.
The
occasional
washing
of
the
system
with
methanol
was
found
to
be
a
very
efficient
method
for
avoiding
the
risk
of
air
bubbles
being
trapped
on
the
inner
walls
of
the
tubes
and
flow
cell.
2.4.
Sample
preparation
of
ascorutin
tablets
Five
tablets
were
accurately
weighed
and
crushed
to
a
powder.
The
amount
equivalent
to
one
tablet
was
weighed,
dissolved
by
gentle
warming
in
methanol,
transferred
to
a
25-mL
volumetric
flask,
and
the
volume
was
filled
up
with
water.
The
solution
was
then
filtered
through
a
Whatman
no.
41
paper
filter
to
separate
the
insoluble
sample
matrix.
Then
a
0.25
or
0.5
mL
of
this
solution
was
M.k.E.A.
Al-Shwaiyat
et
al.
/
Journal
of
Pharmaceutical
and
Biomedical
Analysis
149
(2018)
179–184
181
Fig.
1.
Schematic
diagram
of
a
SI-LAV
manifold
for
the
simultaneous
determination
of
Asc
and
rutin.
transferred
to
a
25
mL
flask;
the
volume
was
filled
up
with
water,
and
the
resulting
solution
was
analysed
as
described
previously.
3.
Results
and
discussion
3.1.
Colour
reaction
of
18-MPA
with
rutin
and
ascorbic
acid
Under
appropriate
conditions,
the
reaction
of
18-MPA
with
suf-
ficiently
strong
reducing
agents
is
fast
and
accompanied
by
the
formation
of
highly
coloured
heteropoly
blue
(HPB).
The
influence
of
the
solution
pH
on
the
formation
of
heteropoly
blue
produced
in
the
reaction
between
rutin
or
Asc
and
18-MPA
was
studied
in
batch
conditions
begins
to
interact
with
18-MPA
at
sig-
nificantly
lower
pH
values
than
rutin
due
to
the
higher
reduction
potential
(Supplementary
Fig.
S-1).
At
pH
<
4
the
reaction
rate
is
slow,
while
at
pH
>
4.0
the
reaction
takes
less
than
one
minute
to
complete
The
reaction
between
rutin
and
18-MPA
pro-
ceeds
more
slowly.
About
90%
of
the
heteropoly
blue
is
formed
after
2
min,
but
even
after
15
min
absorbance
continues
to
grow
slowly.
At
pH
>
5
oxidation
of
rutin
by
18-MPA
begins
to
contribute
to
the
formation
of
heteropoly
blue.
Thus,
the
optimum
pH
range
for
the
selective
determination
of
Asc
in
the
presence
of
rutin
was
found
to
be
between
4
and
5.
Simultaneous
and
complete
oxida-
tion
of
both
Asc
and
rutin
occurs
in
the
pH
range
from
7
to
9.
At
pH
>
9,
a
concurrent
reaction
of
the
destruction
of
18-MPA
becomes
noticeable.
These
features
were
used
as
the
basis
for
developing
a
method
of
simultaneous
determination
of
Asc
and
rutin.
It
is
based
on
measurement
of
the
absorbances
of
HPBs
formed
at
pH
4.7
and
7.4.
Asc
is
then
determined
directly
from
the
absorbance
mea-
sured
for
the
solution
with
pH
4.7,
while
for
calculation
of
the
rutin
concentration
the
difference
of
the
absorbances
measured
for
the
two
above-mentioned
samples
is
used.
It
was
found
that
only
a
small
excess
of
reagent
is
required
for
the
complete
oxidation
of
both
analytes.
Therefore,
the
optimal
concentration
of
18-MPC
was
proposed
to
be
of
0.15
mmol
L
−1
,
providing
an
acceptably
wide
concentration
range
for
the
determination
of
rutin
or
Asc.
It
was
found
by
investigation
of
the
reaction
between
18-MPA
and
reducing
agents,
including
rutin
and
Asc,
that
the
calibration
curves
obtained
often
showed
appreciable
non-linearity.
Two
fun-
damental
reasons
were
established
as
being
responsible
for
such
an
undesirable
phenomenon.
The
change
in
the
ratio
of
reducing
Fig.
2.
Absorption
spectra
of
heteropoly
blues
obtained
by
reduction
of
18-MPA
with
rutin.
C
Rutin
,
mol
L
-1
:
30
(1),
20
(2),
6
(3),
2
(4);
C
18-MPA
=
0.16
mmol
L
-1
;
pH
=
7.4;
l
=
1
cm;
t
=
30
min.
agent
to
18-MPA
leads
to
marked
changes
in
the
spectra
of
the
HPBs
formed
The
isobestic
point
at
approximately
920
nm
is
present
in
the
spectra
plotted
as
a
dependence
of
molar
absorp-
tivity
of
HPB
versus
wavelength.
The
observed
phenomenon
might
be
explained
by
the
existence
of
an
equilibrium
between
two
types
of
HPBs
in
the
studied
system.
In
the
great
excess
of
18-MPA,
one-
electron
HPB
is
formed
in
accordance
with
the
following
equation:
P
2
Mo
VI
18
O
62
6
−
+
H
2
P
2
Mo
VI
16
Mo
V
2
O
62
6
−
=
2P
2
Mo
VI
17
Mo
V
O
62
7
−
+
2H
+
Reducing
the
residence
time
of
HPB
in
the
RC
leads
to
an
increase
in
the
nonlinearity.
The
calibration
graph
plotted
using
4
min
of
reaction
time
had
a
noticeable
curvature
(R
2
=
0.988).
This
curva-
ture
was
diminished
with
increasing
reaction
time
and
for
20
min
the
calibration
curve
obtained
was
perfectly
linear
(R
2
=
0.9995)
(Supplementary
Table
S-1).
Investigation
of
the
spectra
of
HPBs
obtained
for
different
ratios
of
Folin-Ciocalteu
(FC)
reagent
to
reducing
agent
(
showed
that
the
shape
of
the
spectra
is
systematically
changed
and
strongly
dependent
on
the
ratio
of
reagent
to
reducing
agent.
The
band
max-
imum
is
shifted
hypsochromically
from
740–760
nm
to
∼650
nm
by
decreasing
this
ratio
at
high
concentrations
of
the
analyte.
No
182
M.k.E.A.
Al-Shwaiyat
et
al.
/
Journal
of
Pharmaceutical
and
Biomedical
Analysis
149
(2018)
179–184
Fig.
3.
Absorption
spectra
of
heteropoly
blues
obtained
by
reduction
of
Folin-
Ciocalteu
reagent
with
rutin.
C
Rutin
,
mol
L
-1
:
2
(1);
10
(2),
20
(3),
40
(4),
100
(5),
200
(6);
l
=
1
cm;
t
=
30
min.
Preparation
of
the
solutions.
Aliquot
of
rutin
solution,
0.3
mL
of
FC
reagent
[40]
and
3
mL
of
20%
Na
2
CO
3
were
mixed
in
a
25
mL
volumetric
flask.
The
flask
was
then
filled
with
distilled
water
to
the
mark.
Table
1
Determination
of
Asc
and
rutin
in
different
synthetic
binary
mixtures.
Amount
added
(
mol
L
−1
)
Recovery
(%
±
Rutin
Asc
Rutin
Asc
20
20
100.2
±
1.5
99.4
±
1.2
10
20
98.4
±
1.7
99.2
±
2.1
5
20
97.2
±
3.1
100.5
±
1.8
7.5
60
104
±
4
101.0
±
1.3
3.5
60
107
±
8
99.3
±
1.4
a
Mean
and
standard
deviation
for
five
determinations.
Table
2
Influence
of
some
interfering
species
on
the
determination
of
10
mol
L
−1
of
rutin.
Species
Tolerable
concentration
(mmol
L
−1
)
NaNO
2
0.1
Na
2
SO
3
1
KI
50
Glucose,
saccharose
20
Citric,
oxalic,
tartaric
acid
30
Salicylic,
sulfosalicylic,
acetylsalicylic
acid
10
Caffeine
40
Thiamine
chloride
2
Folic
acid
1
Phenol
8
Thymol
0.1
Pyridoxine
hydrochloride
0.5
isobestic
point
was
found
in
such
spectra.
Such
dependence
of
the
spectrum
shape
on
the
reagent/analyte
ratio
makes
obtaining
accurate
results
of
analysis
using
FC
reagent
for
the
determina-
tion
of
individual
reducing
compounds
questionable.
Therefore,
this
approach
is
not
applicable
for
the
simultaneous
determina-
tion
of
rutin
and
Asc.
In
addition,
it
was
found
that
the
oxidation
of
Asc
and
rutin
occurs
simultaneously
at
all
the
pH
values
studied.
3.2.
Optimization
of
the
SI-LAV
manifold
parameters
In
the
paper
conditions
were
found
for
the
deter-
mination
of
Asc
and
rutin
in
mixtures
using
the
SIA
method.
The
developed
procedure
is
characterized
by
good
sensitivity
and
preci-
sion.
Nevertheless,
a
preliminary
study
showed
that
several
factors
may
negatively
influence
the
determination
of
low
quantities
of
rutin
in
the
presence
of
a
great
excess
of
Asc.
The
analytical
signal
for
rutin
is
obtained
as
a
difference
of
absorbances
measured
for
two
samples
acidified
to
different
pH
and
carried
out
through
the
entire
developed
procedure.
Such
difference
is
especially
sensitive
to
the
errors
arising
by
subtracting
two
nearly
equal
numbers.
Due
to
dispersion,
the
absolute
values
of
the
absorbances
obtained
in
the
SIA
method
are
approximately
three-times
less
than
in
steady
conditions
and
less
reproducible.
In
addition,
the
strong
Schlieren
effect
caused
by
the
high
concentration
of
18-MPC
greatly
dete-
riorates
the
SIA
signal
at
low
and
to
a
lesser
extent
at
higher
concentrations
of
analyte
taking
the
above
circumstances
into
consideration,
it
was
desirable
to
develop
another
method
for
the
simultaneous
determination
of
Asc
and
rutin
having
adequate
accuracy,
precision
and
sensitivity.
It
was
proposed
to
supplement
the
SIA
configuration
with
an
external
RC
in
order
to
achieve
better
analytical
parameters
in
the
analysis
of
ascorutin
tablets.
Conducting
the
mixing
process
in
an
RC
guarantees
the
effective
and
rapid
mixing
of
the
reactants.
In
addition,
the
residence
time
for
slow
reactions
can
be
reduced
by
carrying
out
the
mixing
and
equilibration
of
the
reactants
in
an
external
RC.
The
use
of
air
bubbling
instead
of
mixing
with
a
mag-
netic
bar
simplifies
the
configuration
of
the
manifold.
The
magnetic
stirrer
does
not
belong
to
the
standard
equipment
of
SIA
instru-
ments
and
needs
additional
programming
and
interfacing
making
the
building
of
the
overall
flow
system
more
cumbersome.
By
using
the
external
RC,
the
optimization
of
the
key
variables
is
greatly
simplified,
because
the
parameters
of
the
analytical
method
found
under
batch
conditions
can
be
employed
practically
without
any
changes.
Only
a
corresponding
scaling
of
the
volumes
of
reagent
and
sample
was
undertaken.
The
volume
of
reagent
used
in
the
SI-
LAV
method
was
lower
by
two
orders
of
magnitude
than
in
the
batch
procedure,
i.e.
20
L
of
0.15
mM
18-MPC
instead
of
2
mL.
In
this
respect,
it
is
noteworthy
that
4
mM
concentration
of
18-MPC
was
used
in
the
preceding
SIA
procedure
3.3.
Linearity,
accuracy,
and
precision
of
the
method
Three
calibration
curves
were
constructed,
including
two
cali-
bration
graphs
for
Asc
at
pH
4.7
and
7.4,
and
the
calibration
curve
for
rutin
at
pH
7.4.
The
analytical
signal
for
rutin
was
calculated
from
the
difference
between
the
absorbance
measured
for
the
sample
acidified
to
pH
7.4
and
the
absorbance
for
Asc
recalculated
from
pH
4.7
to
pH
7.4
using
the
appropriate
calibration
graphs.
Under
the
optimized
conditions
at
the
solution
pH
7.4,
the
calibration
curves
were
linear
over
the
concentration
ranges
from
5
×
10
−7
to
4
×
10
−5
M
(0.3–24
mg
L
−1
)
and
from
1
×10
−6
to
8
×10
−5
M
(0.2–14
mg
L
−1
)
for
rutin
and
Asc,
respectively.
The
cor-
responding
linear
regression
equations
of
the
calibration
plots
calculated
for
the
rutin
at
pH
7.4
and
Asc
at
two
pHs
(7.4
and
4.7)
were
the
following:
A
=
0.014
±
0.003
+
(2.93
±
0.02)
×
10
4
×
C
rutin
(r
2
=
0.9997,
n
=
10);
A
=
(1.39
±
0.05)
×
10
4
×
C
Asc
(r
2
=
0.9991,
n
=
7),
and
A
=
(1.31
±
0.07)
×
10
4
×
C
Asc
(r
2
=
0.9984,
n
=
7),
respectively.
The
concentration
of
analytes
is
expressed
in
mol
L
−1
.
Absorbance
was
measured
at
920
nm
in
a
flow
cell
with
an
optical
path
length
of
20
mm.
The
limit
of
detection
was
calculated
as
three-times
the
ratio
of
the
standard
deviation
of
the
intercept
of
the
slope
of
the
cali-
bration
plot
(i.e.
LOD
=
3
×
s
a
/slope)
and
the
limit
of
quantification
as
10-times
this
ratio
(LOQ
=
10
×
s
a
/slope).
The
limit
of
detection
for
rutin
was
found
to
be
0.2
moL
L
−1
(0.13
ppm),
and
the
limit
of
quantification
was
0.6
moL
L
−1
.
The
limit
of
detection
calculated
for
Asc
for
the
data
obtained
at
pH
4.7
was
equal
to
0.5
moL
L
−1
(0.09
ppm).
The
detection
limit
of
the
proposed
method
is
compa-
rable
with
that
of
the
SIA
method
in
the
latter
method,
large
systematic
errors
occur
when
using
the
lower
part
of
the
graduation
graph
due
to
the
Schlieren
effect.
Under
the
optimized
conditions,
the
throughput
was
calculated
as
15
h
−1
,
with
negligible
carryover.
The
accuracy
and
precision
of
the
method
were
evaluated
by
analysing
a
series
of
standard
binary
mixtures
of
Asc
and
rutin
At
favourable
ratios
of
Asc
to
rutin
(lower
than
4:1)
the
M.k.E.A.
Al-Shwaiyat
et
al.
/
Journal
of
Pharmaceutical
and
Biomedical
Analysis
149
(2018)
179–184
183
Table
3
Results
for
the
determination
of
Asc
and
rutin
trihydrate
in
ascorutin
by
the
proposed
and
the
reference
methods
(mg/tablet
±
,
n
=
5,
95%
confidence
level).
Producer,
weight
of
a
tablet
Claimed
value
Found
by
the
proposed
method
Found
by
the
reference
method
Asc
Rutin
Asc
Rutin
Asc
Zentiva,
Czech
republic,
0.5
g
100
20
98.1
±
2.1
21.1
±
1.8
101.4
±
1.5
20.7
±
0.4
Kyiv
vitamin
factory,
Ukraine,
0.33
g
50
50
49.2
±
0.8
50.6
±
1.6
50.3
±
0.7
49.4
±
1.2
a
The
drug
is
rutin
trihydrate
20
mg
and
ascorbic
acid
100
mg
in
1
tablet.
The
other
ingredients
are:
sodium
citrate,
lactose
monohydrate,
potato
starch,
castor
oil,
gelatine,
corn
starch,
Sepifilm
752.
b
1
tablet
contains
ascorbic
acid
50
mg
and
rutin
trihydrate
50
mg.
The
other
ingredients
are:
sugar,
potato
starch,
calcium
stearate,
talc.
c
Determination
with
2,6-phenolindophenol.
d
Determination
with
AlCl
3
.
precision
of
the
determination
of
both
substances
varied
in
the
range
from
1
to
2%.
At
higher
ratios,
the
precision
of
rutin
deter-
mination
progressively
worsened,
and
at
more
than
20-fold
molar
excess
of
ascorbic
acid
to
rutin,
the
relative
standard
deviation
exceeded
10%.
3.4.
Interference
study
and
application
The
interfering
action
of
typical
interferents
for
the
reaction
between
18-MPC
and
various
reducing
agents
or
other
substances
has
already
been
studied
at
pH
4–5
pH
7.4
of
the
studied
polyphenols
more
or
less
completely
react
with
18-MPA
at
pH
7.4
while
no
interferences
were
found
for
reducing
sugars,
salicylic
acid
and
its
derivatives,
caffeine,
oxyacids
and
common
excipients
(sodium
chloride,
EDTA,
magnesium
stearate,
lactose,
talc
and
starch)
at
[interferent]/[rutin]
ratios
much
higher
than
those
found
commonly
in
pharmaceuticals
Along
with
Asc,
thiols
show
strong
interference.
The
reactions
with
folic
acid,
thiamine
and
monophenols
become
important
at
comparatively
high
concentrations
of
these
compounds
in
a
strongly
basic
solu-
tion.
The
proposed
method
was
applied
to
the
determination
of
Asc
and
rutin
in
ascorutin
tablets.
The
results
of
analyses
of
pharmaceu-
ticals
obtained
using
the
proposed
and
reference
methods
agreed
well
with
the
claimed
values
of
producers
in
all
instances,
thus
confirming
the
accuracy
and
suitable
precision
of
the
developed
method
(
In
addition,
the
content
of
the
drugs
in
ascorutin
was
evaluated
by
standard
methods.
Asc
was
determined
with
2,6-dichlorophenolindophenol
and
the
aluminium
chloride
method
was
used
for
the
determination
of
rutin
comparison
of
the
results
obtained
by
the
proposed
and
the
reference
methods
confirm
the
validity
of
the
developed
method.
4.
Conclusions
A
simple,
sensitive,
green
and
accurate
SI-LAV
method
has
been
developed
for
the
simultaneous
determination
of
two
active
sub-
stances
in
ascorutin.
The
developed
method
demonstrates
suitable
precision
even
at
a
large
interferent-to-analyte
ratio
by
determi-
nation
of
rutin
in
the
presence
of
up
to
a
20-fold
molar
excess
of
Asc.
Only
water
was
used
as
a
carrier
and
solvent,
and
no
previous
separation
of
the
components
was
required.
The
flexibility
of
the
SIA
system
was
significantly
improved
by
integration
with
an
external
RC,
which
allowed
for
elimination
of
the
Schlieren
effect
and
increased
the
precision
as
well
as
the
sensitivity
of
the
determination.
Reagent
and
sample
consump-
tion
and
the
volume
of
effluents
were
maintained
at
the
lowest
levels
possible
and
are
thus
in
accordance
with
the
principles
of
green
chemistry.
The
concentration
of
the
reagent
is
two
orders
of
magnitude
lower
than
that
used
under
batch
conditions.
The
spectrum
of
HPB
formed
in
the
reaction
of
FC
reagent
or
18-MPC
with
reducing
agents
depends
on
the
ratio
of
analyte
to
reagent.
This
phenomenon
is
caused
by
the
formation
of
different
reduced
forms
of
heteropoly
blues
coexisting
in
such
solutions.
By
using
18-MPC
as
reagent,
measurement
of
the
absorbance
at
the
wavelength
corresponding
to
the
isobestic
point
allows
strictly
lin-
ear
calibration
graphs
to
be
obtained
and
systematic
errors
by
the
determination
of
individual
reducing
agents
or
their
mixtures
to
be
avoided.
FC
reagent
cannot
be
recommended
for
using
as
reagent
for
the
determination
of
individual
species
due
to
the
absence
of
isobestic
point
in
the
corresponding
spectra
of
HPBs.
Acknowledgments
A.
Vishnikin
gratefully
acknowledges
the
financial
support
pro-
vided
by
the
Slovak
Academic
Information
Agency.
This
work
was
financially
supported
by
the
Scientific
Grant
Agency
of
the
Min-
istry
of
Education
of
the
Slovak
Republic
and
the
Slovak
Academy
of
Sciences
(VEGA
grant
1/0253/16).
References
[1]
[2]
[3]
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
184
M.k.E.A.
Al-Shwaiyat
et
al.
/
Journal
of
Pharmaceutical
and
Biomedical
Analysis
149
(2018)
179–184
[15]
[16]
[17]
Document Outline
- Simultaneous determination of rutin and ascorbic acid in a sequential injection lab-at-valve system
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