Jordan Journal of Chemistry Vol. 8 No.2, 2013, pp. 103-112

103

JJC

Visible Spectrophotometric Determination of Ganciclovir in its Pharmaceutical Formulations using Quinalizarin Reagent

Usra I.S. Al-Neaimy∗a and Amal M. S.Al-Delymi*b a College of Veterinary Medicine, Mosul University, Mosul, Iraq. b Chemistry Department, College of Education, Mosul University, Mosul, Iraq.

Received on Jan. 30, 2013 Accepted on June 3, 2013

Abstract

A simple, rapid and sensitive spectrophotometric method for the determination of Ganciclovir

was developed. The method is based on the proton transfer reaction of drug with quinalizarin

reagent in aqueous solution to form violet product which shows maximum absorbance at 560

nm. Beer's law was obeyed in the concentration range 1-20 µg.mL-1 with an average recovery

(accuracy) of 99.02% and precision (RSD) of less than 2.0%. The molar absorbtivity was

8.93x103 l.mol-1.cm-1 with LOD of 0.21 µg.mL-1 and LOQ of 0.71 µg.mL-1. The proposed method

was further applied to the determination of the drug in pharmaceutical formulations as an

injection and capsule and the results were comparable with the certified values of

pharmaceutical formulations.

Keywords: Ganciclovir; Spectrophotometric; Quinalizarin.

Introduction Ganciclovir (GCV) or 9-(1, 3-dihydroxy-2-propoxymethyl)guanine, (Figure 1),is

an acyclic nucleoside analogue of 2-deoxyguanosine that inhibits replication of herpes

viruses. It is used for the prevention of cytomegalovirus (CMV) disease in organ or

bone marrow transplant recipients and in HIV-infected individuals who are at risk of

developing CMV disease. GCV is a white crystalline powder with a molecular formula

of C9H13N5O4 and a molecular weight 255.23 g/mol [1, 2].

Figure 1: The chemical structure of ganciclovir

Various analytical techniques were used for the determination of GCV in

biological fluids or pharmaceutical formulations which include high-performance liquid

chromatography,[3] capillary electrophoresis,[4] differential pulse and square wave

∗ Corresponding author: e- mail: dr.am53@yahoo.com

104

voltammetry[5]. These techniques require sophisticated instruments and expensive

reagents and involve several manipulation steps and derivatization reactions.

However, spectrophotometric techniques continue to be the most preferred methods

for routine analytical work due to their simplicity and reasonable sensitivity, along with

significant economical advantages. Literature survey revealed few spectrophotometric

methods which have been developed for the determination of GCV in bulk drug and its

pharmaceutical formulations such as the reaction of GCV with p-dimethylamino

cinnamaldehyde, [6] a charge transfer reaction of GCV (n- electron donor ) with several

σ and π acceptors[7] and first order derivative spectroscopy.[8] GCV also was

determined at 253 nm in 0.1 N HCl and at 266 nm in 0.1 N NaOH [9], the oxidation of

the drug by different inorganic oxidants in acidic medium[10] and the reduction of

potassium permanganate by ganciclovir in alkaline medium[11].

Quinalizarin has been frequently utilized as an analytical reagent in

pharmaceutical analysis. It has been used for the colorimetric determination of

piroxicam and tenoxicam,[12] albendazole,[13] azithromycin,[14] cephalexin [15], aliphatic

amines. [16]

In this work quinalizarin reagent was used for the spectrophotometric

determination of ganciclovir via proton transfer reaction in aqueous solution.

Experimental

Apparatus A digital double beam Shimadzu UV-1650 spectrophotometer with 1- cm glass

cells used for all spectral and absorbance measurements. The pH of the solutions

was measured by HANNA (pH211) Microprocessor pH meter.

Reagents

All chemicals used were of the highest purity available which were provided by

BDH and Fluka companies. Ganciclovir was obtained from (European Directorate for

the Quality of Medicines & HealthCare).

Standard ganciclovir (1000 µg.mL-1) solution

A stock standard solution of 1000 µg.mL-1 GCV was prepared in distilled water

and diluted further with distilled water to get a working solution of 100 µg.mL-1.

Quinalizarin (QA) 2x10-4 M solution This solution was prepared by dissolving 0.0136 g of quinalizarin in a adequate

quantity of absolute ethanol and diluted to the mark in a 250 mL volumetric flask with

the same solvent. The solution was stable for at least one month.

0.1% w/v surfactant

(Cetavlon, cetyltrimethylammonium bromide and sodium dodecyl sulphate ) in

water.

105

Buffer solutions pH 10 [17]

Carbonate buffer : a solution of sodium carbonate (0.05 M) was adjusted to pH 10 with

a pH meter by the addition of adequate amount of 0.1 M HCl.

Phosphate buffer : 0.05 M NaH2PO4, 0.1 M HCl.

Borate buffer : 0.025 M Na2B4O7. 10 H2O, 0.1 M NaOH.

Procedure for calibration

To a series of 10 mL volumetric flasks, increasing volumes of ganciclovir

working standard solution were transferred to cover the range (1-20) µg.mL-1 in final

dilution. Quinalizarin solution (4 mL of 2x10-4 M) was added. The solution was diluted

to the mark with distilled water. The absorbance was measured at 560 nm after 5

minutes at room temperature against the blank solution which was prepared in a

similar way.

Assay procedure for dosage forms

1-Injection:

Several vials of cymevene (IV) from Roche, that contain 500 mg ganciclovir,

were obtained. The contents of two vials were mixed, and an amount equivalent to 500

mg of the component was weighed and dissolved in distilled water and filtered then

completed to the mark in a 100 mL volumetric flask. From the above solution 20 mL

was pipette into a 100 mL volumetric flask and the volume was made up to the mark

with distilled water. Further dilution was made with distilled water to get the

concentration of 100 µg.mL-1.The solution was proceeded as described under

procedure for calibration.

2-Capsule:

The mixed contents of 10 capsules (Lovir from Oubari Pharma – Aleppo – Syria)

were accurately weighed. An amount equivalent to 250 mg of the drug was dissolved

in sufficient quantity of distilled water and filtered then the volume was made up to 100

mL with distilled water. From the above solution 40 mL was pipette out into a 100 mL

volumetric flask and the volume was made up to the mark with distilled water. Further

dilution was made with distilled water to get the concentration of 100 µg.mL-1. The

solution was prepared as described under procedure for calibration.

Results and discussion Absorption spectrum

Ganciclovir reacted with quinalizarin in aqueous solution to give violet colored

complex with maximum absorption at 560 nm, whereas the reagent blank gives

maximum absorption at 480 nm (Figure 2).

O

E

o

d

I

E

6

o

t

Fq

Figure 2: A

Optimization

Effect of pH

The pH

of 2 mL of

different buff

t was found

Effect of the

The ef

6 mL) of the

of GCV and

the addition o

Figure 3: Eff

quinalizarin c

Absorption sp

of condition

H of ganciclo

2 x 10-4 M

fers of pH 10

that these bu

volume of qu

ffect of quina

reagent, wh

following the

of 4 mL of th

fect of (2x10-

complex

0

0.1

0.2

0.3

0.4

Absorba

nce

pectra of: (A)

(B)

s

ovir was mea

M quinalizarin

0, (i.e. borate

uffers decrea

uinalizarin:

alizarin reage

ich was add

e general pro

e reagent (F

-4 M) QA volu

0 2

106

quinalizarin

) reagent bla

asured, and f

n the value o

e, carbonate

ase the abso

ent was inves

ed to an aliq

ocedure. The

Figure 3).

ume on the a

2 4

Volume of 

with gancicl

ank.

found to be

of pH decrea

e and phosph

orbance .

stigated by ta

quot of soluti

e better abso

absorbance o

6

QA., mL

ovir (10 µg m

11. After the

ased to 10, t

hate) were p

aking various

on containin

orption was o

of 10 µg mL-

8

mL-1) against

e addition

therefore

prepared.

s amounts (1

g 10 µg. mL

observed wit

1 GCV-

t

1-

L-1

th

107

Effect of time and temperature:

The reaction time was determined by following the color intensity at room

temperature and in thermostatically controlled water bath adjusted at 40 and 50 C°.

The experiment showed that the color of the solution developed after 5 minutes and

the absorbance remained stable for more than two hours. All studied conditions were

optimized at room temperature (30 ±1 C°), at which the best color intensity was

obtained (Figure 4).

Figure 4: Effect of time and temperature on the absorbance of

10 µg mL-1 GCV – quinalizarin complex Effect of surfactant

Different types of surfactants, including cetavlon, cetyltrimethylammonium

bromide (CPC) and sodium dodecylsulphate (SDS) were tested to enhance the

absorption of the colored product. Unfortunately none of them improve the absorption

and therefore they were excluded from this study.

Quantification

Under the experimental conditions, standard calibration graph for ganciclovir

was constructed by plotting the absorbance versus concentration (Figure 5). Beer's law

plot was obeyed in the concentration range (1-20) µg.mL-1 for GCV with a correlation

coefficient, molar absorbtivity, regression equation were given in Table 1. In order to

determine the accuracy and precision of the proposed method, the recovery (R %) and

relative standard deviation (RSD %) for three different concentrations of drug were

also calculated and recorded in table 2.

0

0.1

0.2

0.3

0.4

0 20 40 60 80 100 120 140

Absorba

nce

Time , (min.)

30 C°

40 C°

50 C°

108

Figure 5: Calibration graph of ganciclovir

Table 1: Quantitative parameters for the complexation of GCV with Quinalizarin

Values Parameter

560 λmax (nm)

1-20 Beer's law limit (µg.mL-1)

8.93x103 Molar absorptivity (l.mol-1.cm-1)

Regression equation(Y)*

0.035 Slope (a)

0.002 Intercept(b)

0.998 Correlation coefficient (r)

0.21 LOD ( µg.mL-1)

0.71 LOQ ( µg.mL-1)

*Y=ax+b, where x is the concentration of GCV in µg.mL-1 and Y is the absorbance.

Table 2: Accuracy and precision of the method.

RSD* (%) Recovery* (%) Found

(µg. mL-1) Taken

(µg. mL-1)

± 1.07 99.00 4.95 5

±0.71 98.80 9.88 10

±0.46 99.26 14.89 15

* Average of six determinations

Effect of interferences

To check the selectivity of the method, 10 µg. mL-1 of ganciclovir was

determined using the recommended procedure in the presence of foreign compounds

that are usually present in pharmaceutical formulations. The results didn't show any

interfering effect on the present method which indicate that the method was selective

(Table 3).

y = 0.035x + 0.002R² = 0.998

00.20.40.60.8

0 5 10 15 20 25

Absorba

nce

Concentration ,µg . mL-1

109

Table 3: Effect of excipients for assay of ganciclovir

Recovery* (%) ± SD of 10 µg mL-1 GCV per µg excipient added Excipient

3000 2000 1000 500

101.44±1.18 102.85±0.34 99.98±0.45 101.45±0.92 Starch

101.99±0.67 101.55±0.55 98.54±0.43 102.43±1.54 Magnesium stearate

103.11±1.32 103.31±0.69 98.67±0.24 102.65±0.81 Croscarmellose sodium

101.49±0.24 102.32±0.12 99.76±0.84 99.54±0.98 Microcrystalline cellulose

* Average of three measurements.

Stoichiometry and reaction mechanism

In the present work the stoichiometric ratio for the reaction of ganciclovir with

quinalizarin was investigated through applying the continuous variation (Job's)

method[18], with using equimolar solutions of each (2x10-4 M). It was found that

ganciclovir forms a product with quinalizarin in the ratio of 1:1, as it is evident in figure

6. The probable reaction mechanism based on the reported method is given in

scheme1.

Figure 6: Continuous variation plot for reaction of ganciclovir with quinalizarin under

the optimum conditions.

Scheme 1: Proposed reaction mechanism for assay of GCV

0

0.05

0.1

0.15

0.2

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1

Volume ratio of GCV (VGCV/VGCV+VQuin) 

Absorba

nce

e

a

a

c

w

o

4

T

A

p

s

f

Accor

estimated b

amounts (2x

amount of q

conditional st

Kc=1-α

α =Am-A

where Kc is t

of the comple

4 indicate tha

Table 4: Stab

Volume ogan

0

0

1

Analytical a

The p

pharmaceutic

standard add

from interfere

Figure 7: S

rding to the

y comparing

10-4 M) of ga

quinalizarin

tability const

α/ α2C

As/Am

the stability c

ex which is e

at the produc

bility constan

f (2x10-4 M) onciclovir

.5 mL

.7 mL

.0 mL

pplication proposed m

cal formulatio

dition proced

ences as sho

Standard add

results desc

g the abso

anciclovir an

reagent (A

tant of the co

constant, α th

equal to the c

ct was stable

nt of ganciclo

of Coganci

1

1.4

2

method was

ons. The val

dure. The res

own in table

dition graph

110

cribed above

orbance of

nd quinalizari

Am) using 1

omplex was c

he dissociati

concentration

e.

ovir - quinaliz

onc.oficlovir(M)

x10-5

4x10-5

2x10-5

s applied

idity of the m

sults showed

5, and figure

of GCV in ph

e, the appare

a solution

in (As) to one

0 mL calibr

calculated by

on degree an

n of drug. Th

zarin comple

As Am

0.07 0.1

0.132 0.15

0.148 0.18

to the det

method was c

d that the pr

es 7 and 8.

harmaceutica

ent stability

containing s

e containing

rated flask.

y applying th

nd C is the c

he results sho

x

m α

2 0.416

53 0.137

83 0.191

termination

confirmed by

roposed met

al formulation

constant wa

stoichiometri

an excessiv

The averag

e equation:

concentration

own in table

Average K(l.mol-1)

1.57x106

of GCV i

y applying th

thod was fre

n (injection)

as

ic

ve

ge

n

Kc

in

he

ee

T

Pharmacedosage f

Cymevene (I

Lovir(Capsu

*Average of

C

p

T

Anal

Linear

Molar abs

Limit of

Limit of Q

Ty

Comp

Analy

Figure 8: S

Table 5: Ass

Ceutical form

njection)

2r ule)

three determi

Comparison

Table

preparations

Table 6: Co

ytical parame

λmax (nm)

Temp (°C)

Reagent

r range (µg. m

sorptivity 1. cm-1)

Detection (LOmL-1)

Quantification µg. mL-1)

ype of reaction

position of the

ytical applicat

Standard add

say and recov

Certified value

500mg/vial

250mg/capsule

nations

of methods

6 displays

using the pr

mparison of

ter

mL-1)

( l. mol-

OD/ µg.

(LOQ/

n

dye

ion

dition graph

very of GCV

AmounGCV ad(µg. m

e

510

5e 10

the results

resent metho

the present

Present meth

560

30

Quinalizarin

1-20

8.93103

0.21

0.71

Proton transf

1:1

Injection ancapsules

111

of GCV in ph

V in pharmace

Drnt of dded L-1) Pres

met

4.89.8

4.8

9.7

s for determ

od and those

method with

hod

n

fer

d

harmaceutica

eutical dosag

ug content fou(mg)

Stanaddimet

sent thod

5.087 10.88

5.181

10.75

mining the

e found in lite

literature me

Ref. (6)

524

40

p-dimethylamcinnamaldehy

10-50

1.175 × 10

0.425

4.60

Schiff baseformation

1:1

Capsules

al formulatio

ge forms.

und

Prme

dard ition hod

97.405 98.802

96.211

97.529

GCV in ph

erature. [6,11]

ethods

mino yde po

03

e

n (capsule)

Recovery*(%

adresent ethod

140±0.23 180±0.36

120±0.44

150±0.57

harmaceutica

Ref.(1

610

25

otassium perm

2-100

4.59x1

0.21

0.72

Oxidation R

1:2

Injection and

%) ± SD

Standard ddition method

101.00±0.29 100.20±0.81

102.20±0.64

102.90±0.82

al

1)

manganate

0

03

eaction

capsules

d

112

It is evident from this table that the present method has good sensitivity

compared to some recently published methods on GCV determination. Conclusion

The proposed method is very simple, rapid and sensitive. The method does not

involve the use of complicated sample preparation. Low value standard deviation

shows that the method is precise, whereas high percentage of recovery shows that the

method is free from interference of the excipients used in the formulations, also the

reported method requires neither buffer solution nor solvent extraction. Therefore the

method can be useful in routine quality control analysis.

References [1] Sweetman S. C., "Martindale: The Complete Drug Reference", 34th Edn, Pharmaceutical

Press,London, (2005) pp. 635-637. [2] Katzung B. G., ´´ Basic and Clinical Pharmacology´´. 9th Edn., Mc Graw Hill, Singapore,

(2007) pp.806-808. [3] Boulieu, R., et al., J.Chromatogr., 1991,567, 481-484. [4] Saleh, S. ; Hempel, G., Electrophoresis,2006,27, 2439-2443. [5] Uslu, B., et al., Anal. Chim. Acta, 2005,537, 307-313. [6] Sarsambi, P. S., et al., Int. J. ChemTech Res.,2010, 2, 282-285. [7] Gouda, A. A., Talanta,2009, 80, 151-157. [8] Sarsambi, P. S,et al.,Int. J. PharmTech. Res., 2010,2, 1264-1268. [9] Sarsambi, P. S., et al., J. Ind. Council Chem.,2010, 27,202-204. [10] Gouda,A.A. ; Amin, A.S., Lat. Am. J. Pharm.,2011, 30 (2),334-341. [11] AL-Neaimy, U.I. ; Hamdon,E.A., Raf. j. Sci., 2012,23(4), 93-104. [12] Amin, A.S., J. Pharm.Biomed.Anal.,2002,29, 729-736. [13] Kamel,M.S., et al., Journal of Applied Sciences Research,2008, 4(10): 1242-1248. [14] De Paula, C.E.R.,et al., J. Braz. Chem. Soc., 2010, 1-8. [15] Almeida, V.G.K. ; Cassella,R.J., Quim. Nova, 2010, 33( 4), 914-919. [16] Al-Ghabsha, T.S.; Al-Delymi, A.M.S., J. Educ. & Sci.,2009, 22 (1). [17] Perrin D.D. and Dempsey B., ״Buffers for pH and ion control״, Chapman and Hall Ltd,

London (1974). [18] Hargis L.G., "Analytical Chemistry, Principels and Techniques", Prentice – Hall Inc., New

Jersey. (1988) pp. 424 – 427.