Electrochemical Detection of Zanamivir Using Gold and Gold-Modified Boron Doped Diamond Electrode

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Muhammadiyah University of Surakarta, Solo Centrat lava tndonesia )anuary 10 th,

2Ats

Proceeding

International

Conference

"Current Breakthrough in Pharmacy Matetials and Analyses"

/

Nurwaini ef aL, (ed) Surakarta : Muhammadiyah University'Press, 20Ls

xxi, 153 pages

ISBN : 97 8- 602-7 0 429 -9 -5

Pharmacy

Chairman of Editors

Team of Pharmaceutical Technology

Setyo Nurwaini, M.Sc.

Anita Sukmawati, Ph.D., Apt, Suprapto, M.Sc., Apt.

Erindyah Retno Wikantyasning, Ph.D., Apt.

Setyo Nurwaini, M.Sc.

Dedi Hanwar, M.Si., Apt. Dr. Muhammad Da'i, M.Si., Apt. Broto Santoso, M.Sc., Apt.

Dra. Nurul Mutma'inah, M.Si., Apt.

Zaklry Cholisoh, M.Clin. Pharm., Ph.D., Apt. Arifah Sri Wahyuni, M.Sc., Apt.

Tanti Azizah Sujono, M.Sc., Apt.

Zakky Cholisoh, M.Clin. Pharm., Ph.D., Apt. Arifah Sri Wahyuni, M.Sc., Apt.

Dra. Nurul Mutma'inah, M.Si., Apt.

Ratna Yuliani, M.Biotech.St.

Azis, Saifudin, Ph.D., Apt.

Ratna Yuliani, M.Biotech.St.

Azis, Saifudin, Ph.D., Apt.

Team of Chemical Analyses

Team of Pharmacy Community and Clinic

Team of Pharmacological Study

Team of Herbal Medicine

'feam of Microbiology Pharmacy

Copyright

@20L5

'.

Copyright in compilers and reserved

Design cover: Pubiication and Documentation Team

Layout: Secretaris and IT Team

Published by:

Muhammadiyah University Press

Universitas Muhammadiyah Surakarta

Il.A.Yani Pabelan Tromol Pos I Kartasura SurakartaSTL02 Telp. (+62 27 L) 7 L7 4L7 -L72, E-mail: muppress@yahoo.com

Proceeding International Conference

"Current Breakthrough in Pharmacy Materials and Analyses"

ISBN : 97 8-602-7 0429 -9 -5

TABLE OF CONTENTS

LIST OF PARTICIPANT NON

PRESENTERS...

_...ix

LIST OF ORAL

PRESENTERS...

_...xv

LIST OF POSTER

PRESENTERS

_...xviii

B.PHARMACEUTICAL

TECHNOLOGY

_{... 1}

B-OO2

DEVELOPMENT OF _{PARTICULATE FORMULATION, LOADING PROCEDURE AND}

NANO.SCALE CHAMCTERIZATION

OF PLGA

NANOPARTICLES INCOORPOMTING ROOT EXTMCT OF _{Pueraria lobata (Wild) Ohwl} Mardiyantol*, Neudrt

Achimz

_...1

8-005

FORMULATION OF _Jatropha Multifida Linn. LATEX AS TOPICAL DosAGE FORM AND WOUND HEALING STUDY IN SWISS WEBSTER MICE Amilar, Fetri Lestarir, Abdul

Rahmant

_...,...g

8-008

EFFECT OF USE VIRGIN COCONUT

OIL

_[VCO) AS EMOLTENT ON PHYSICAL PROPERTIES AND STABILITY OF VITAMIN C IN TRANSPARENT SOAP Ms. Fatimah Kasorr., Anita

Sukmawatit...

...16

B.O1O

.

INCREASING SOLUBILITY AND DISSOLUTION OF GLIMEPIRIDE THROUGH CO. CRYSTALLIZATION METHOD Fitrianti Darusmapr*, Sundani N Soewandhiz, Rachmat

Mauludins...

_...2s

8-012

SYNTHESIS

OF

POLY(METHACRYLIC

ACID)

_CROSS-LINKED

BY

_SILVER NANOPARTICLES FOR pH-RESPONSIVE NANoSENSoR Erindyah R. Wikantyasningl*, Bunga Saviti-ir, BrotoSantosol,

Suprapto

_...33

C-CHEMICAL

ANAIYSES.."...

...

_{...!...r ...42}

C.OO1

ELECTROCHEMICAL DETECTION OF ZANAMIVIR USING GOLD

AND

GOLD-MODIFIED BORON DOPED DIAMOND ELECTRODE

wulan Tri wahyunil,2*, Ivandini rribidasari A1, Endang

Saepudin

...q2

C.OOz

OPTIMIZATION OF HPLC CONDITION FOR DETERMINATION OF VITAMIN A AND

Dg IN PHARMACEUTICAL PREPARATIONS

Engrid |uni Astutit, Mochammad Yuwonoz, Muhamad

Zainuddinz

...4g

Proceeding International Confe rence

"current Breakthrough in pharmacy Materials and Analyses,,

ISBN : 97 8-602-7 A429 -9 -S

e

ETECTROCHEMICAT DETECTION OF ZANAMIVIR USING GOI,D AND GOLD-MODIFIED

BORON DOPED DIAMOND ETECTRODE

Wulan

Tri

Wahyunir,z.,

Ivandini Tribidasari

41, Endang Saepudint

rDeportment _{of Chemistry,} Faculty of Mathematics and Natural Sciences, University

of

I ndon e si a, D epok I ndonesia

z _{Department of Chemistry, Faculty of l4athematics and Natural Sciences, Bogor} Agricultural

U niversity, B og or Indonesia

.E-mail _{: wulantriw!@ipU.ac.id}

Abstract

Zanamivir

is a

neuraminidase

inhibitor

approved

by

WHO

for

treatment and

prophylaxis of influenza virus A and B. Quantitative analysis of zanamivir is important to

monitor

its

content

in

medicine and aquatic environment.

This

study addressed the

electrochemical detection

of

zanamivir using

gold and

gold-modified

boron

doped

diamond (BDD) electrodes. The measurement was conducted using cyclic voltammetry method. Logarithmic curve was consistently observed from measurement of zanamivir in wide range of concentration on three electrodes. Linear calibration curve of zanamivir was

obtained

in

the concentration range

of

L

x

10-s

-

1

x

10-a

mol/L

(r'

-

0.969) on gold

electrode,5x10-6-1x10'+mol/L(rz=0.990)onAu-BDDelectrode,and1xL0-6-1x1.0-+

mol/L

(r,

-

0.998) on AuNPs-BDD. Meanwhile, the detection

limit

estimated to be L.19 x

1.0-6,'1..49

x

1,0-s, and 2.29

x

10-6 molfL, respectively

for

gold, Au-BDD, and AuNPs-BDD

electrode. The precision of zanamivir measurements on three electrodes was good with

%RSD < 2.5 o/o.

Keywords : Diamond, Electrochemical, Gold, Voltammetry, Zanam ivir.

INTRODUCTION

Zanamivir (Figure 1) is a neuraminidase inhibitor approved by WHO for treatment and

prophylaxis of influenza virus A and B. This compound prevents the release and spread of the

neurly formed virion through blocking the active site of neuraminidase. Zanamivir therapy was

reported

to

be more effective

for

influenza B treatment compare

to

oseltamivir (fcrmer

neuraminidase inhibitor) [1]. The use of zanamivir was rose as-oseltamivir resistant virus was

reported [2]. Examination of zanamivir content in commercial drugs should be conducted in

order

to

ensure that the patients get the appropriate.treatment dosage. lmproper dosages

lead

to

resistance effect of the viruses. Furthermore, the concentration of zanamivii' in the

aquatic environment also important

to

be

monitored due

to

rising

content

by

human

Proceeding International Conference

"Current Breakthrough in Pharmacy Materials and Analyses"

ISBN : 978-602-7 0429-9-5

excresions and improper disposal _[3]. Undesired

high

levels

of

antivirats

'in

the

aquatic

environment which contact with the viruses are potentially cause resistance.

Several analysis methods was reported

for

zanamivir determination, such as LC-MS

[4,5J and detection using mercury drop electrode _[5]. These techniques are effective for

zanamivir measurement, otherwise LC-MS needs several types of chemicals and sophisticated

instrument. Meanwhile, detection using mercury drop electrode was avoided because of safety reason. Due to this reasons, a alternative method for zanamivir measurement need to

be developed.

HO

Figure L Molecular structure of Zanamivir.

Electrochemical method

for

zanamivlr measurement is prospective

to

be devetoped

since

it

is

simple,

fast,

and reagentless.

ln

this work,

electrochemica! measurement of

'zanamivir

using gold and gold modified boron clopecl diarnond electrode (BDD) was developed.

BDD was used as working electrode due to itslow background currents, stability, and wide

potential window in aqueous _{solution [7].} Meanwhile, gcld used

for

BDD modification to

enhanced its performace in zanamivir sensing.

MATERIALS AND METHODS

Materials

Zanamivir (Tokyo Chemical Industry Co. Ltd.), KAuCI+.4HzO (\ffako Chemicals),

allylamine (Tokyo

chemical

Industry co.

Ltd.),

Na:csHsoz, KzHpo+, KHzpo+, HzSo+, CEHgBOa, hydrogen, oxygen, methane, silicon

rlafer,

diamond powder, 2-propanol, Gold

electrode, Ag/AgCl as reference eiectrode, platinum as counter electrode, and high

purity

vrater

with

maximum conductivity

of

18

MO obtained

from

Simply-Lab

water

system

(DIRECT-Q 3 UV, Millipore).

HN

)-NH-//

HN

Proceeding International Conference

"Current Breakthrough in Pharmacy Materials and Analyses"

ISBN : 97 8-602-7 0429 -9 -s

Methods

Preparation of Working Electrode

Boron doped diamond (B/C atomic ratio

of

1:L000) were deposited on Si [100) wafers in microwave plasma-assisted chemical vapor deposition (MPCVD) system (ASTeX

Corp.). BDD was electrochemically polarized at 3 V (vs. SSCE)

in

O.1

mol/L

HzSO+ for 20

min

to

obtain oxygen terminated BDD. Au-BDD was prepared through electrochemical deposition in equal volume of 2 mM HAuCI+.4HzO and HzSO+ 0.5 mM mixture at 0.2 V (vs.

SSCE) for 100 _{min, detail preparation are describe elsewhere [8]. Meanwhile,} AuNPs-BDD

was obtained by photochemical reaction using allylamine followed by chemical reaction

with

AuNPs. The AuNPs prepared according to _{Turkevich and Frens method [9, 10].} XPS

were used to characterize the Au-BDD and AUNPs-BDD electrode

prior

to use as working electrode.

Electrochemical Measurement of Zanamivir

Electrochemical measurement

of

zanamivir

was

performed

in a

single compartment cell

with

three electrodes. A platinum

wire

was used as counter electrode and silver/silver chloride electrode (SSCE) used as reference electrode. Working electrode Au-BDD and AuNPs-BDD was pressed against the bottom of the electrochemical cell by an

O-ring

with

the geometric area approximately 0.L25 cmz. Phosphate buffer (PBS) 0.1 M

was used as

the

supporting electrolyte. Cyclic voltammetry measurements

of

various conce.ntrations

of

zanamivir

in

PBS 0.L

M

was performed at

a

scan rate

of

L00 mVs-t.

Precision

of

zanamivir measurement

was

expressed as percentage

relative

standard

deviation

[%

RSD) which obtained from nine replicates analysis of three concentration level of zanamivir.

RESULTS AND DISCUSSION

Hydrogen terminated BDD

with

0.L o/o _{boron has successfully prepared by MPCVD}

system. Raman spectra (Figure 2a) shows an intense peak at 1.332 cm-l which confirmed

that carbon sp3 of diamond have already deposited on Si wafer _[7] and the broad peak at

500

cm'l

expected

to

be boron doped

on

diamond

film

_{U7,721. Diamond crystals} with

approximately 3 pm in size were covered the silica wafer (Figure 2b).

Proceeding International Conference

"Current Breakthrough in Pharmacy Materials and Analyses"

ISBN : 97 8-602-7 0429 -9 -S

160000

?120000

o

'6 _aoooo

C

o

c

-

40000 [image:7.595.57.536.59.316.2]

400

800

1200 1600 2000 Raman shift / cm-l

Figure 2 Raman spectra (a) and SEM profile (b) of 0.1 0/o BDD.

Gold was electrochemically deposited on BDD surface. Two peaks observed at83.2

eV and BT ev are attributable to Au a,fi/z a1f, Au 4F/z,respectively (Figure 3a). Au particles expected to be physically deposited at BDD surface

without

any chemical bonding since

there was

no

significant

shift of

C 1s peak

at

Au-BDD electrode. On

the

other hand,

modification of the BDD surface

with

AuNPs was initiated by photochemical reaction of

BDD surface with allylamine followed by chemical reaction of amine-terminated BDD with

AuNps. Amine functional groups facilitated the chemical bonding

of

AuNPs

with

BDD

surface. XpS spectra confirm that AuNPs has successfully deposited on amine-terminated

BDD. XpS peaks of Au 4frlz and 4f,p was detected at binding energies of 84 and BB eV,

respectively (F igure 3b).

Figure

4

shows cyclic voltammograms (CV)

of

0.1

M

PBS

pH

7

obtained

at

a

potential scan rate

of

100 mVs-r in the absence and

in

the presence of Zanamivir at Au electrode. There is no new oxidation or reduction peak observed in potential range of 0 V to +L.SO V. However, the CV showed that the presence of Zanamivir leads to the decrease

of oxidation and reduction peak currents

of

gold. As proposed

in

our

previous report, Zanamivir was adsorp on gold electrode and reduces the

direct

contact between gold surface and waterfoxygen

[8].The

condition leads

to

the

decrement

of

oxidation and

reduction of gold surface.This afford the chance for Zanamivir measurement using gold

and gold modified BDD.

Proceeding International Conference

"Current Breakthrough in Pharmacy Materials and Analyses"

ISBN : 978-602-7 0429-9'5

(a) c1s

3CC

[image:8.595.88.539.61.241.2] [image:8.595.186.399.301.459.2]

tlinding Energy _/ eV

Figure 3 XPS spectrum of Au-BDD (a), oxidized BDD, allylamine modified BDD, and AuNps modified BDD (b).

Zan 1.5 x l0r M

-0.5 0.0 0.5

E/V(vs.SSCE)

Figure 4 Cyclic voltammogram of 0.1 M PBS pH 7 in the absence and in the presence of

Zanamivir at Au electrode.

Logarithmic curue was consistently observed from measurement of zanamivir in wide

range

of

concentration on three electrodes (data not shown). Meanwhile,linear calibration

curve of zanamivir was obtained in the concentration range ofL x 1-0-s - L x 1O-+mol/L on gold

electrode,5x10-6-1x10-amol/LonAu-BDDelectrode,andLxL0-e-1x1g-smol/Lon

AUNPs-BDD. The _{linear calibration curve could be developed based on gold oxidatiop and}

reduction peak current. Otherwise, calibration curve developed from gold reduction peak current not always give excellent determination coefficient. Figrrre 5 shows the CV of 0.1 M PBS pH 7

in

the presence _of Zanamivir at various concentrations _{and calibration curve}

obtained

from

measurement

on

each

electrode.

While

Au

electrode shows higher

sensitivity _{than Au-BDD and AuNPs-BDD electrode, higher determination coefficient can}

be achieved using Au-BDD _{and AuNPs-BDD electrodes. The detection}

limit

_{estimated to be}

Proceeding International Conference

"Current Breakthrough in Pharmacy Materials and Analyses"

ISBN : 97 8-602-7 0429 -9 -5

I I t'

1.19

x

LO-6,L.49

x

10-s, and2.29

x

10-6 molfL, respectively

for

gold, Au-BDD, and AuNPs-BDD electrode. Furthermore, the precision of the current responses was investigated for

nine

consecutive measurements.

The

high

precision

af

Zanamivir measurement was reflected

in

the

relative standard deviations (RSDs), which are less than 2.5o/o at each

electrodes. The summery of the analytical performance of Zanamivir at Au and Au-BDD was presented in Table 1..

(a) _{,;,!-!'1 ,t:} (b)

- -':ls

g-*_.14%

+ _=-i 3il3x + ii5.F"? i'- =,' *r'r.

't:i n

Conc:ntrrtion cf Zrnanrivirl PII

r=---..-i L-3 : r, J,

..:

J rt ,) :i

Co ncentr.3tio n of Zr n'rrn ivi r I pld

Au (a,b), Au-BDD (c, dJ, and AuNPs-BDD (e,

₀

electrode.

Proceeding lnternational Conference

"Current Breakthrough in Pharmacy Materials and Analyses"

ISBN : 97 8-602-7 0429 -9 -5

1| i=.:...r-l;=r

A 3z:,3: i:- ==;{

f E C, L L = .J' .:r_,, o o-J

-t !-.i{ i:l :.4 = -:r

l: .jr lF,'lA r,l :.: :.:

E _{'V :,'s.} SSC Ei

47

ll) r

f{ {:r

S{ih

--t---

- i[--. __.,{-_.

{

(ej

,.

-.:

s(

=

-i:

-i. 0

-: ,l

1 - 'z{--... l: 1(8.

lr

=,'V:vE SSC:EI

{ E o L L f,-.J .:r G G,

o-

6--+..-;

4

n.

ConcEntration of Zanarnivirl Pll -t

Table 1 The summary of the analytical performance _{of Au, Au-BDD, and AuNps-BDD}

Electrode

Au-BDD AuNPs-BDD

Linearity ranee [M 1 x 10-s -1 x 10-r

5x10-o-LxL0-+

_{1x1.0'o-1.x1.0-s}

Determination coefficient (Rz1

Oxidation peak

Reduction oeak

Limit of Detection (M) Oxidation peak

Reduction oeak

Precision of measurement n ₌

e (%RSD)

Oxidation peak

Reduction peak

0.990 0.990

0.946 0.998

1..25 x L0's

L.L9 x 10-o

1..53 x L0-s

1.49 x L0-s

1.89 x 10-s

2.29 x 70's

CONCLUSION

Electrochemical detection of Zanamivir

in

0.1 _{M phosphate buffer solution (pBSl} has been successfully investigated at gold (Au) and gold-modified boron-doped diamond

(Au-BDD and AuNPs-BDDJ electrodes. Measurements

of

_{Zanamivir based}

on

_reduction

peak current provide better performance than the oxidation peak. Performance of

AuNps-BDD electrode _{generally better than other two electrodes.}

REFERENCES

1.

Kawai N, Ikematsu H,

Iwaki

N, Maeda

T,

Kanazawa H, Kawashima

T,

Tanaka O,

Yamauchi S, Kawamura K, Nagai T et al. 2OOB. A comparison of the effectiveness of

za'namivir and oseltamivir for the treatment of influenza A and B. _f . Infect. 56: SI-ST.

2.

Azuma

T,

Nakada

N,

Yamashita

N,

Tanaka H.2A1,2. Synchronous dynamics of

observed

and

predicted values

of

anti-influenza drugs

in

environmental waters

during

a

seasonal influenza outbreak, Environ. Sci. Technol. 46: 2g73-L2gB1.

3.

Cunningham

VL,

Binks SP, Olson Ml.2AO9. Human

health

risk

_{assessment from}

the

presence

of

human

pharmaceuticals

in

the

aquatic

_{environment. Regul.}

Toxicol. Pharmacol. 53: 39-45.

4.

Baughman TM, Wright WL, Hutton KA. 2007. _{Determination of zanamivir in rate and}

monkey plasma

by

positive

ion

hydrophilic interaction

chromatography IHILIC)/tandem mass spectrometry. _J. chromatogr B. g52: s0s

-

511.

5.

Ge J, [,iu F, I{olmes EH, Ostrander GK,

Li

_QX.}OLZ Aqueous

normal

phase Iiquid

chromatography

coupled

with

tandem

time-o?-flight quadrupole

_mass

spectrometry

for

deterrnination

of

zanamivir in humah _{serum. |. Chromatogr} B.

58

-

62,

6.

Skrzypek S. _{2010. Electrochemical Studies of the Neuraminidase Inhibitor Zanamivir}

and its Voltammetric Determination in Spiked Urinb. Electroan al.22:2339-2346.

7.

Fujishirna A, Einaga Y, Rao TN, Tryk DA. _{Diamond Electrochemistry. First ed. Elsevier} Science. Tokyo: 2005.

8.

Wahyuni WT, Ivandini TA,

fiwanti

_{PK, Saepudin E Gunlazuardi}

f,

_Einagay..

Z0l4.

Electrochemical Detection of Neuraminidase based on Zanamivir lnhibition using

Gold-Proceeding International Conference

"current _{Breakthrough in Pharmacy Materiars and Anaryses"}

ISBN : 97 8-602-7 0429 -9 -5

Modified Boron-Doped Diamond Electrode. Research _{Report. lndonesia: University of} lndonesia.

Turkevich

f,

Stevenson PC,

Hiller

J. 1951.

A

study

of the

nucleation and growth

processes _{in the synthesis of colloidal gold. Discuss. Faraday Soc. 11: 55-75.}

Frens

C.

1'973- _{Controlled nucleation}

for the

_regulation

of

the

particle

_{size in} monodisperse gold suspensions. Nat. phys. Sci.24'1.: zo-zz.

ACKNOWTEDGMENT

Directorate

of

higher

education Republic

of

Indonesia

and

Department of

Chemistry Keio University, _fapan.

OPTIMIZATION OF HPLC CONDITION FOR DETERMINATION OF VITAMIN A AND D3

IN PHARMACEUTICAT PREPARATIONS

Engrid _f uni Astutit, _{Mochammad Yuwonoz, Muhamad Zainuddinz}

t _{Pharmacy Deportment,}

Il niversity of Muhammadiyah Molang

zFaculty _{of Pharmacy, Airlangga IJniversier Surabaya}

Abstract

The

purpose

of

the present study was

to

achieve the HPLC condition

for

the

separation

of

vitamin

A

and Dg

in

_{pharmaceutical formulations.}

The

_{optimization of} method was carried

out by

investigating the effect

of

HPLC _{conditions, namely mobile}

phase composition,

flow

rate, packing

of

column

and

analytical wavelength

on

peak symmetry, tailing factor, resolution and retention time reproducibility of the analytes of

interest.

A

good

simultaneous separation

of

vitamin

A

and

De

in

pharmaceutical fprmulations was obtained using a _{chromolith@ column Rp-1ge (100 mm x 4.6 mm) with}

methanol L00 o/o _{as mobile phase at flow rate}

of L ml/min. The detector was set

at

265.g

nm and total analysis time was 12 min. The system suitability test demonstratecl good

resolution (Rs>5),

tailing

factor

<

2, theoretical plate

>

2000 and selectivity

factoi

>1.

Relative standard deviation (RSD) of retention time was 0.1 1.0/o for vitamin A and o.A7o/a

for vitamin D3, whilst RSD of peak area _{'was 0.09o/o} and 0.99o/o for vitamin A and vitamin

D3, resp€ctively.

Keyword : vitamin A and D3, HpLC, Method optimization.

INTRODUCTION

Vitamins are

a

group

of

organic compounds

thrt

,..,

in

very

_{small amounts,} essential _{for normal metabolism, growth and development, and regulation}

of cell function. Thirteen vitamins are recognized in human nutrition which may be conveniently classified

into two groups according to their solubility : as water-soluble (B-complex and C) and fat-soluble (A, D, E and K) _t1l.

Proceeding International Conference

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9.

10.