VALIDATION OF A QUANTITATIVE DETERMINATION METHOD

OF DICLOFENAC FOR IN VITRO BIOEQUIVALENCE

EVALUATION OF TRANSDERMAL GEL PREPARATIONS

S. I. Guseva,1 M. V. Karlina,2 O. N. Pozharitskaya,2 A. N. Shikov,1, 2 and N. M. Faustova3

Translated from Khimiko-Farmatsevticheskii Zhurnal, Vol. 44, No. 1, pp. 46 – 49, January, 2010.

Original article submitted January 1, 2009.

A quantitative determination method of diclofenac for studying the bioequivalence of transdermal gel prepa-

rations of this drug has been validated. It was established that the proposed method is precise, reproducible,

and linear in a certain concentration range. The release of diclofenac from transdermal gels with different vis-

cosities and from the reference preparation was studied using the paddle-over-disk technique. Drug release

from the transdermal gel preparation was modeled using various mathematical laws including the Higuchi

law, first-order kinetics, cube root, and Weibull function. An analysis of the experimental data showed that the

release of diclofenac from gels proceeds by diffusion and obeys the Higuchi law. It was found that the rate of

drug release from the gel depends significantly on the viscosity. Similarity coefficients were calculated in or-

der to compare diclofenac dissolution profiles and to determine their equivalence. It was concluded that ex-

perimental samples of diclofenac gel with medium and high viscosity were equivalent to the reference prepa-

ration.

Key words: diclofenac, transdermal gels, bioequivalence testing in vitro.

Diclofenac (2-[2, 6-dichlorophenyl)amino]benzeneacetic

acid) is a nonsteroid anti-inflammatory preparation (NAIP)

(Fig. 1) [1]. Diclofenac is offered on the drug market as the

free acid and the sodium and potassium salts [2].

Diclofenac is widely used in medical practice in various

medicinal forms for both systemic effects and local therapy

[3 – 7].

Gels are a promising medicinal form for local anti-in-

flammatory therapy. As a rule, the presence of alcoholic sol-

vents used to prepare the gels provides rapid penetration of

the drug through the skin [8]. The gel composition also in-

cludes components that ensure the active ingredient passes

through the dermis and underlying tissue [9].

Bioequivalence testing (pharmacokinetic equivalence) of

drugs is the principal type of medical and biological control

of generic drugs that have the same medicinal form and con-

tent of active ingredients as the corresponding original drugs

[10]. In vitro tests, in particular the dissolution test, can be

used as alternatives to the classical studies for testing

bioequivalence.

Our goal was to validate a quantitative determination

method of diclofenac in a dissolution medium and to com-

pare the diclofenac release profiles from test gels of different

viscosity and the reference preparation diclonat P.

EXPERIMENTAL PART

We used test samples of diclofenac gel of low, medium,

and high viscosity and the reference preparation diclonat P

(Pliva Hrvatska d.o.o., Croatia). The diclofenac content in

each sample was 1%. Hydroxypropylcellulose at different

concentrations was used as the viscosity modifier in test

samples.

Diclofenac release from the test preparations used the

paddle-over-disk method according to USP 30 and an

Erweka series DT 600 tester (Germany) for checking the sol-

ubility [11].

The conditions were dissolution medium temperature, 32

± 1°C; paddle rotation rate, 50 rpm; dissolution medium, wa-

ter; dissolution medium volume, 150 mL. Samples (3 mL)

were taken at given time intervals (5, 10, 15, 30, 45, 60, 90,

43

0091-150X/10/4401-043 © 2010 Springer Science+Business Media, Inc.

Pharmaceutical Chemistry Journal Vol. 44, No. 1, 2010

1Mechnikov St. Petersburg State Medical Academy, St. Petersburg,

195067, Russia.2

St. Petersburg Institute of Pharmacy, St. Petersburg, 195067, Russia.3

Adaptogen Corporation, Interregional Medical Center, St. Petersburg,

Russia.

120, 180 min) and filtered through a 0.45-�m filter. The dis-

solution medium volume was restored using water.

Quantitative analysis of diclofenac used a spectrophoto-

metric method at 275 nm. Measurements were made on a

PharmaSpec 1700 UV-Vis spectrophotometer (Shimadzu, Ja-

pan).

The diclofenac dissolution process was modeled using

the following methods: Higuchi equation, first-order kinetic

equation, cube-root law, and Weibull model [12].

Dissolution rate constants were calculated by

least-squares methods [13].

Dissolution profiles were compared by calculating the

similarity coefficient f2

[14, 15] using the formula:

fn

R Tj j

j

n

2

2

1

0 5

50 11

100� � �

�

�

�

�

�

�

�

�

�

�

�

�

��

�

�

�

��

�

log | |

.

�

,

where n is the number of time points and Rj

and Tj

are the

percent dissolved compound from the preparation at each

time point j.

RESULTS AND DISCUSSION

Validation of quantitative determination method

Process validation is an important part of a quality assur-

ance and control system and is obligatory in practice for

quality manufacture of a medical product.

A spectrophotometric method was chosen to analyze

diclofenac in the dissolution medium because it was accu-

rate, rather fast, and could combine qualitative and quantita-

tive analyses of the principal component. The choice of de-

tector wavelength (275 nm) was based on the specific maxi-

mum of the substance in the UV spectrum.

The determination method of diclofenac in the dissolu-

tion medium was validated in accordance with recommenda-

tions given for methods used in the Dissolution test

44 S. I. Guseva et al.

N

HOOC

Cl

ClH

Fig. 1. Diclofenac structural formula (C14

H11

Cl2NO

2).

0.5

0.4

0.3

0.2

0.1

0

220 250 300 350

�, nm

275 nm

1

2

Fig. 2. Spectra of placebo solution (1 ) and diclofenac gel in water

(2 ).

TABLE 1. Validation Parameters of Diclofenac Determination

Method

Parameter Value

Linearity and analytical range, �g/mL 2.57 – 20.24

Regression equation (Y = bX + a) Y = 0.0343X + 0.0051

Correlation coefficient r 0.9970

Relative standard deviation, RSD, % 0.17

Standard deviation for b (Sb) 0.00068

Standard deviation for a (Sa) 0.0092

Accuracy, % 97.5 – 102.5

Precision

Intraday precision, RSD, %

2.53 �g/mL 2.36

10.1 �g/mL 1.41

20.2 �g/mL 0.58

Interday precision, RSD, %

2.53 �g/mL 2.77

10.1 �g/mL 1.46

20.2 �g/mL 0.60

Limit of detection, LOD, �g/mL 0.50

Limit of quantitation, LOQ, �g/mL 1.67

TABLE 2. Determination Coefficients (R2) of Mathematical

Models and Dissolution Rate Constants for Diclofenac Preparations

Model Diclonat P

Diclofenac gel

low viscositymedium vis-

cosityhigh viscosity

Higuchi equa-

tion

0.9689 0.8464 0.9841 0.9679

First-order ki-

netic equation

0.9412 0.7608 0.9780 0.9233

Cube-root law 0.6280 0.4267 0.6976 0.6144

Weibull distri-

bution func-

tion

0.9888 0.8926 0.9961 0.9793

Dissolution rate

constant,

min– 1

0.7915 1.0856 0.6517 0.9095

[16 – 18]. The validation method parameters were the speci-

ficity, linearity and analytical range of the method, accuracy

(or correctness), limit of detection, and limit of quantitation.

Table 1 lists the validation parameters for the method.

Spectra of a model mixture of excipients included in the

preparation (placebo) were recorded in order to check the

specificity of the method. It was found that the excipients did

not interfere with the diclofenac determination in the dissolu-

tion medium (Fig. 2).

The resulting values of the statistical criteria for the ex-

amined concentration range satisfied the requirements [18].

Therefore, the validated method can be used for the Dissolu-

tion test.

The stability of diclofenac in the dissolution medium was

also evaluated. It was shown that the solutions were stable

for 1 d at room temperature and for 2 d at 8 – 10°C.

Diclofenac release rate

Topical application was modeled using the pad-

dle-over-disk method that is applicable for transdermal me-

dicinal forms [11]. Distilled water was chosen as the dissolu-

tion medium. A total of 12 determinations were made for

each preparation. Figure 3 shows the results.

Analysis of the data in Fig. 3 showed that diclofenac re-

lease from all studied samples was practically linear for 60

min with the exception of the sample with low viscosity. This

was related to the destruction and/or dissolution of the gel

matrix into which the substance was incorporated. On aver-

age, about 78.5 ± 6.0% of the active ingredient dissolved into

the medium after 60 min. Then, the process slowed and

reached 88.4 ± 4.7% after 3 h. The dissolution rate constants

calculated by least-squares methods (Table 2) showed that

the dissolution was fastest from the diclofenac gel of low vis-

cosity (1.0856 min– 1

). The dissolution rate of the reference

preparation and test gel samples with high viscosity differed

insignificantly at 0.8459 � 0.0815 min– 1

(RSD 9.6%).

Various mathematical functions were used to describe

the diclofenac dissolution process for the transdermal appli-

cation model. Linearization of the release curves showed that

the Weibull distribution function and Higuchi equation were

most suitable for describing the release of diclofenac from

the preparations (Table 2).

The results led to the conclusion that diclofenac release

from the test gels of medium and high viscosity and from

diclonat P occur through diffusion and obey the Higuchi law

[19].

Similarity coefficients (f2) were calculated in order to

compare dissolution profiles of diclofenac and to determine

their equivalence. These reflect the difference between two

curves over all time points. The dissolution profiles can be

considered similar if the f2

values are from 50 to 100

[14, 15]. The similarity coefficients for the studied prepara-

tions were:

diclonat P—low viscosity diclofenac gel, 35.55

diclonat P—medium viscosity diclofenac gel, 55.15

diclonat P—high viscosity diclofenac gel, 63.89.

Based on these calculated similarity coefficients, it was

concluded that the experimental samples of diclofenac of

medium and high viscosity were equivalent to the reference

preparation diclonat P.

ACKNOWLEDGMENTS

The work was supported financially by ZAO Natur

Produkt Interneshnl (Russia).

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Validation of a Quantitative Determination Method of Diclofenac 45

100

90

80

70

60

50

40

30

20

10

0

0 30 60 90 120 150 180

t, min

4

1

3

2Dic

lofenac

rele

ase,%

Fig. 3. Diclofenac release profiles from test diclofenac gels of low, medium, and high viscosity and diclonat P reference preparation (M � m,

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46 S. I. Guseva et al.