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Contents

lists

available

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.

E-mail

address:

vishnikin@hotmail.com

(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.

background image

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

electrochemical

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

background image

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

background image

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

background image

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]

spectrophotometric

[5]

pharmaceuticals

[6]

[7]

spectrophotometric

[8]

electrochemical

nanotubes–chitosan

[9]

[10]

D. ˇSatínsk ´y,

[11]

spectrophotometric

[12]

molybdophosphate

[13]

spectrophotometric

[14]

background image

184

M.k.E.A.

Al-Shwaiyat

et

al.

/

Journal

of

Pharmaceutical

and

Biomedical

Analysis

149

(2018)

179–184

18-molybdodiphosphate

[15]

[16]

[17]

Document Outline

Simultaneous determination of rutin and ascorbic acid in a sequential injection lab-at-valve system