Journal of the Chinese Chemical Society, 2005, 52, 1049-1054 1049

Simultaneous Spectrophotometric Determination of Benzyl Alcohol and

Diclofenac in Pharmaceutical Formulations by Chemometrics Method

Jahanbakhsh Ghasemi,a* Ali Niazia and Sirous Ghobadib

a
Chemistry Department, Faculty of Sciences, Razi University, Kermanshah, Iran
b
Biology Department, Faculty of Sciences, Razi University, Kermanshah, Iran

Diclofenac sodium (DS) is a drug with analgesic, antipyretic, and anti-inflammatory properties. It is pres-
ent in numerous pharmaceutical preparations. In injectable forms, it is usually accompanied by benzyl alcohol
(BA) as an excipient, which is used as a blocking anesthetic (4%) and an antiseptic (4-10%). In this work a
spectrophotometric methodology was applied in order to determine benzyl alcohol and diclofenac in inject-
able formulations by applying a multivariate calibration method. By a multivariate calibration method such as
partial least squares (PLS), it is possible to obtain a model adjusted to the concentration values of the mixtures
used in the calibration range. In this study, the concentration model is based on absorption spectra in the
230-320 nm range for 25 different mixtures of benzyl alcohol and diclofenac. Calibration matrix contains
10-95 and 1-50 mg mL-1 for benzyl alcohol and diclofenac, respectively. The root mean square errors of pre-
diction (RMSEP) for benzyl alcohol and diclofenac were 3.0776 and 1.7557, respectively. The proposed
method was validated by using a set of synthetic sample mixtures and subsequently applied to simultaneous
determination of benzyl alcohol and diclofenac in two different pharmaceutical formulations.

Keywords: Benzyl alcohol; Diclofenac; Spectrophotometry; Partial least squares.

INTRODUCTION benzyl alcohol and diclofenac sodium10,11 was achieved by

Diclofenac sodium (DS), sodium salt of [2-(2,6-dichlo-
rophenyl)amino]phenyl]acetic acid, is a potent non-steroidal
anti-inflammatory agent for treatment of rheumatoid arthri-
tis. The pharmacological effects of this drug are thought to be
related to the inhibition of the conversion of arachidonic acid
to prostaglandins, which are the mediators of the inflamma-
tory process.1,2 Diclofenac sodium is usually accompanied by
benzyl alcohol (BA) as an excipient when used in injectable
form.1 Even though benzyl alcohol is present as an excipient,
it is important that its concentration dose not exceed limit val-
ues for each formulation type, since it can produce fatal toxic
syndromes, allergies, and undesirable effects over the ner-
vous system.
Generally, benzyl alcohol is quantitatively determined
by gas chromatography and diclofenac sodium has been de-
termined by a variety of analytical techniques, such as gas
chromatography,3 differential scanning calorimetry,4,5 spec-
trophotometry,6,7 spectrofluorimetry,8 and liquid chromatog-
raphy. 9 Simultaneous determination of benzyl alcohol and
diclofenac sodium in pharmaceutical preparations containing
derivative methods.
Spectral overlap and non-specific irrelevant absorption
affect the interpretation of data for even the simplest-com-
ponent drug systems, leading to variable intercepts on the
absorbance axis and systematic errors in the graphs of ab-
sorbance versus concentration. These drugs present partially
overlapping spectra. Derivative techniques and multivariate
calibration such as partial least squares (PLS) have been de-
vised for the analysis of mixtures with overlapping spec-
tra.12,13 The advantage of multicomponent analysis using par-
tial least squares is the speed of the interest in a mixture, as a
separation step can be avoided.
The theory and application of partial least squares
(PLS) in spectrometry have been discussed by several work-
ers. 12-19 Several multicomponent determinations based on
the application of these methods to spectrophotometric data
have been reported in the literature.20-30 The aim of this pa-
per is to evaluate the possibility of using the partial least
squares method for simultaneous determination of benzyl al-
cohol and diclofenac (Fig. 1), in pharmaceutical formula-
tions.

* Corresponding author. E-mail: jahan.ghasemi@tataa.com

1050 J. Chin. Chem. Soc., Vol. 52, No. 5, 2005
EXPERIMENTAL SECTION
Instrumentation and Software
A Hewlett-Packard 8453 diode array spectrophotom-
eter controlled by a Hewlett-Packard computer and equipped
with a 1 cm pathlength quartz cell was used for UV-vis spec-
tra acquisition. Data acquisition between 230 and 320 nm was
performed with the UV-Vis ChemStation program (Agilent
Technologies), running under Windows XP. A Metrohm 692
pH-meter furnished with a combined glass-saturated calomel
electrode was calibrated with at least two buffer solutions at
pH 3.00 and 9.00.
The data were treated in an AMD 2000 XP (256 Mb
RAM) microcomputer using MATLAB software, version 6.5
(The MathWorks). Partial least squares (PLS) calculus was
carried out in the “PLS-Toolbox”, version 2.0 (Eigenvectors
Company).
Reagents and Standard Solutions
All chemicals used were of analytical-reagent grade.
Subboiling, distilled water was used throughout the work.
Stock solutions (1000 mg mL-1) of benzyl alcohol and diclo-
fenac sodium (Merck) were prepared by direct weighing the
required amount of commercially available reagents. The
composition of the 3 mL commercial injectable form is: di-
clofenac sodium 75 mg and excipients (mannitol, sodium
metabisulfite, benzyl alcohol (4%), propylene glycol, sodium
hydroxide sufficient amount for pH 8.4, and distilled water
apyrogene).
Procedure and Sample Preparation
Known amounts of the standard solutions were placed
in a 10-mL volumetric flask and diluted to the final volume
with deionized water (final pH 8.4). The final concentration
of these solutions varied between 10-95 and 1-50 mg mL-1 for
benzyl alcohol and diclofenac, respectively.
An amount of about 1 mL of the injectable form (men-
tioned above) of diclofenac sodium accurately dissolved in
an appropriate amount of water. The sample was transferred
CH2COONa Cl CH2OH
NH
Cl
Diclofenac Sodium Benzyl Alcohol
Fig. 1. Structural formulate of benzyl alcohol and
diclofenac sodium.
Ghasemi et al.
into a 10-mL calibrated flask and diluted to volume and treated
as described above for the standard drug solution.
RESULTS AND DISCUSSION
Fig. 2 shows the absorption spectra in aqueous solution
of benzyl alcohol and diclofenac separately at pH 8.4. The si-
multaneous determination of benzyl alcohol and diclofenac
in mixtures by conventional spectrophotometric methods is
hindered by strong spectral overlap throughout the wave-
length range. Such a determination could theoretically be fa-
cilitated by the use of partial least squares method.
Conventional univariate calibration
In order to establish the optimal measurement condi-
tions for the joint determination, we used the univariate
method to investigate the effect of experimental variables on
the absorption spectra for the drugs studied. We first checked
the stability of the two analytes in aqueous solution. For this
purpose, we recorded the UV absorbance spectra for solu-
tions of benzyl alcohol and diclofenac as a function of time
and found that the spectra of benzyl alcohol and diclofenac
did not vary appreciably over a period of a few days pro-
vided that the solutions were kept at room temperature in the
dark.
As our aim is to determine benzyl alcohol and diclo-
fenac in injectable formulation, and pH must be 8.4, there-
fore, a sufficient amount of sodium hydroxide is added to all
solutions to maintain pH at 8.4. The temperature was found to
Fig. 2. Absorption spectra of the benzyl alcohol (90 mg
mL-1), and diclofenac (40 mg mL-1).
Determination of Benzyl Alcohol and Diclofenac J. Chin. Chem. Soc., Vol. 52, No. 5, 2005 1051

have no appreciable effect on the spectra of the analytes, and
thus 25 °C was chosen for the subsequent work.
Individual calibration curves were constructed with
several points (Fig. 3), as absorbance vs. analytes concentra-
tion in the range of 10-95 and 1-50 mg mL-1 for benzyl alcohol
and diclofenac, respectively. The wavelengths used to gener-
ate calibration curves were 250 and 276.5 nm for benzyl alco-
hol and diclofenac, respectively. Linear regression results,
line equations, and R2 are also shown in Fig. 3.
Calibration and prediction data sets
Multivariate calibration methods are suitable for the
analysis of large numbers of samples. However, they are not
advisable for the determination of large numbers of analytes
because of the complexity of the calibration matrix. More-
over, the sample preparation and analysis are the most expen-
sive steps in the multivariate calibration procedure.
Multivariate calibration methods such as partial least
squares (PLS) require suitable experimental design of the
standards belonging to the calibration set in order to provide
good predictions. The calibration matrix was designed over
the concentration ranges of 10-95 and 1-50 mg mL-1 for ben-
zyl alcohol and diclofenac, respectively. The calibration ma-
trix used for the analysis is shown in Table 1. For the predic-
tion step, 12 prepared mixtures that were not included in the
previous set were employed as an independent test (see Table
2). To ensure that the prediction and real samples are in the
subspace of the training set, the score plot of first principal
component vs. second was sketched and all the samples are
spanned with the training set scores.
Selection of the optimum number of factors
The optimum number of factors (latent variables) to be

Table 1. Concentration data of the training set for two-

components systems (mg mL-1)
BA DS BA DS BA DS
1.0 10.0 13.3 95.0 37.8 73.8
1.0 31.3 25.5 10.0 37.8 95.0
1.0 52.5 25.5 31.3 50.0 10.0
1.0 73.8 25.5 52.5 50.0 31.3
1.0 95.0 25.5 73.8 50.0 52.5
13.3 10.0 25.5 95.0 50.0 73.8
13.3 31.3 37.8 10.0 50.0 95.0
13.3 52.5 37.8 31.3
13.3 73.8 37.8 52.5

Table 2. Added and found results of the synthetic mixtures of

benzyl alcohol (BA) and diclofenac sodium (DS) by
PLS (mg mL-1)
Added Found Recovery (%)
BA DS BA DS BA DS
25.0 12.0 24.30 11.64 97.0 97.2
74.5 48.0 75.52 50.74 105.7 100.0
24.0 31.5 22.57 32.43 103.0 92.8
63.5 52.0 57.84 55.23 106.2 91.1
95.0 05.0 91.05 05.37 107.4 95.8
95.0 25.5 88.18 27.90 109.4 92.8
83.0 51.0 85.81 50.21 98.5 103.4
40.0 45.0 37.89 46.79 104.0 94.7
37.0 25.0 36.56 25.53 102.1 98.8
48.0 46.5 48.39 47.49 102.1 100.8
42.0 40.0 43.78 38.15 95.4 104.2
Fig. 3. Analytical curve for univariate determination 60.0 50.0 59.73 48.09 96.2 99.6
of benzyl alcohol and diclofenac.
1052 J. Chin. Chem. Soc., Vol. 52, No. 5, 2005 Ghasemi et al.

included in the calibration model was determined by comput- Table 3. Statistical parameters of the optimized matrix using the
ing the prediction error sum of squares (PRESS) for cross- PLS
validation models using a high number of factors (half the Analytes NPC* PRESS RMSEP RSEP (%)
number of total standard + 1), which is defined as follows: Benzyl alcohol 2 3.1741 3.0776 4.9515
Diclofenac 3 1.0945 1.7557 4.4920
PRESS = å ( yi - yˆi ) 2 * Number of principal components.

where yi is the reference concentration for the ith sample and

in Table 2). The results obtained by applying PLS algorithm
y$ i represents the estimated concentration. The cross-valida-
to twelve synthetic samples are listed in Table 2, which also
tion method is employed to eliminate only one sample at a
show the recovery for the synthetic series of benzyl alcohol
time and then PLS calibrates the remaining standard spec-
and diclofenac mixtures. As can be seen, the recovery was
tra.12 By using this calibration the concentration of the sam-
also quite acceptable.
ple left out was predicted. This process was repeated until
each standard had been left out once.
Statistical parameters
One reasonable choice for the optimum number of fac-
For the evaluation of the predictive ability of a multi-
tors would be the number that yielded the minimum PRESS.
variate calibration model, the root mean square error of pre-
Since there is a finite number of samples in the training set, in
diction (RMSEP) and relative standard error of prediction
many cases the minimum PRESS value causes overfitting for
(RSEP) can be used:31
unknown samples that were not included in the model. A so-
lution to this problem has been suggested by Haaland et
å
n
( y pred - yobs ) 2
al.16-18 in which the PRESS values for all previous factors are RMSEP = i =1

compared with the PRESS value at the minimum. The F- n

statistical test can be used to determine the significance of
å
n
( y pred - yobs ) 2
PRESS values greater than the minimum. RSEP (%) = 100 ´ i =1

The maximum number of factors used to calculate the

å(y obs )2

optimum PRESS was selected as 13, and the optimum num-
ber of factors obtained by PLS model are summarized in Ta-
ble 3. In all cases, the number of factors for the first PRESS
values whose F-ratio probability drops below 0.75 was se-
lected as the optimum.
where y pred and y obs are the predicted concentration and the
observed value of the concentration in the sample, respec-
tively, and n is the number of samples in the validation set.
The values of RMSEP and RSEP (%) for benzyl alcohol and
diclofenac are summarized in Table 3.

Determination of benzyl alcohol and diclofenac in syn- Determination of benzyl alcohol and diclofenac in phar-
thetic mixtures maceutical formulations
The predictive ability of the method was determined us- To assess the reliability of the method, two commercial
ing 12 two-component mixtures (their compositions are given pharmaceutical preparations were analysed. Table 4 shows

Table 4. Simultaneous determination of benzyl alcohol (BA) and diclofenac (DS) in

pharmaceutical preparations using the PLS. These formulations contain 75 mg
diclofenac sodium and 120 mg benzyl alcohol
Amount Found Amount Found
Pharmaceutical Recovery (%) Recovery (%)
(mg ± SDa) (mg ± SDa)
preparations
DS DS BA BA
b
Chemidarou 72.2 (± 2.37) 96.7 117.9 (± 3.08) 98.3
Voltarenc 74.1 (± 3.77) 98.8 112.8 (± 3.68) 94.0
a
Relative standard deviation for n = 3
b
Ampoules (from Chemidarou, Ltd.)
c
Ampoules (from Voltaren Ltd.)
Determination of Benzyl Alcohol and Diclofenac
the results obtained as well as the composition of the drug
formulations. The validation of the method has been carried
out by comparing with labeled amounts. As can be seen, the
recovery was quantitative and there were no significant dif-
ferences between the amounts obtained from this method and
labeled amounts. The plots of the prediction concentration
versus actual values are shown in Fig. 4 for benzyl alcohol
and diclofenac (line equations and R2 values are also shown).
CONCLUSION
Based on the results obtained in this work, application
of the PLS to the absorbance spectra produced by benzyl al-
cohol and diclofenac during their simultaneous determina-
tion in pharmaceutical preparations is an effective means for
Fig. 4. Plots of predicted concentration versus actual
concentration for benzyl alcohol and diclofenac
by PLS.
J. Chin. Chem. Soc., Vol. 52, No. 5, 2005 1053
quality control of their manufacture. The ensuring methods
are simple, precise, and affordable; also, they require no com-
plex pretreatment or chromatographic separation of the sam-
ples containing the active ingredients to be determined. The
method is a viable alternative to the existing analytical meth-
ods for routine analyses.
Received October 27, 2004.
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