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2007 The Japan Society for Analytical Chemistry



Comparison of Second Derivative-Spectrophotometric and Reversed-phase

HPLC Methods for the Determination of Prednisolone in Pharmaceutical
D. K. SINGH and Rohan VERMA
Analytical Research Laboratory, Department of Chemistry, Harcourt Butler Technological Institute,
Kanpur-208 002, India

Second derivative-spectrophotometric and high-performance liquid chromatographic methods for the determination of
prednisolone in pharmaceutical formulations have been developed. Determination of prednisolone in tablets was
conducted by using a second-order derivative UV spectrophotometric method at 250 nm (n = 5). Standards for the
calibration graph ranging from 5.0 to 35.0 g/ml were prepared from stock solution. The proposed method was accurate,
with 98% recovery value, and precise, with a coefficient of variation (CV) of 1.38. These results were compared with
those obtained by an exclusively developed isocratic reversed-phase high-performance liquid chromatography (HPLC)
method. An isocratic reversed-phase Bondapak C18 column with acetonitrilecitrophosphate buffer (pH 5; 45:55 v/v)
mobile phase was used and UV detector was set to 241 nm using 11-hydroxyprogesterone as an internal standard.
Calibration solutions used in HPLC were in the range from 2 to 300 g/ml. Results obtained by derivative UV
spectrophotometric method were comparable to those obtained by HPLC method, as far as analysis of variance
(ANOVA) test, Fcalculated, 0.762 and Ftheoretical, 3.89, results were concerned.
(Received April 3, 2007; Accepted June 7, 2007; Published October 10, 2007)

Prednisolone is a member of many corticosteroids that are
employed in a large number of potent drugs. In most of the
cases, these show a very close relationship between biopotency
and structures (Fig. 1). For such reasons, this steroid requires a
careful verification of its purity index in order to avoid loss of
potency or cross contamination. Until now, the evaluation of
related foreign substances has been generally achieved by
means of chromatographic procedures and the derivative
spectrophotometry.1 Actually, in the literature many methods
based on HPLC have also been developed.2 These procedures
have become more generally widespread, in particular for
certain classes of steroids such as corticosteroids, which, owing
to their complex structure, can not be analyzed by GLC
procedure without derivatization. Such methods should provide
sensitivity and selectivity and could be easily adapted for
routine quality control analysis, pre-formulation or similar
studies. There is vast information in the literature about
quantification of prednisolone in pharmaceutical raw material
and dosage forms.3,4 Few analytical procedures based on the
material have also been described for steroids.58 The USP and
BP9,10 described few methods for determination of prednisolone
based on spectrophotometric methods for pharmaceutical
preparations. Most of these methods lack specifity and
selectivity for routine analysis. In the present study, a simple,
economical, accurate and reproducible analytical method, based
on the method reported for determination of 11 To whom correspondence should be addressed.

hydroxyprogesterone in USP, is reported for determination of

prednisolone in raw material and its pharmaceutical dosage
forms. Until now, the development of so many methods has
been generally achieved by means of analytical procedures
based on the HPLC.11,12
spectrophotometric method at 250 nm (n = 5) was used for the
determination of prednisolone in tablets. We found in the
literature no comparison of derivative UV spectrophotometric
studies with the HPLC method for the determination of
prednisolone in tablets has not been reported in literature. For
this reason, it was considered that derivative UV
spectrophotometric method would be applicable in routine
analysis since it does not require any pretreatment procedure.
Furthermore, quantitative determination of prednisolone in
tablets was also performed using an exclusive isocratic
reversed-phase high-performance liquid chromatographic
method (as reference method). The results obtained by the
proposed method were compared with those obtained reference

Fig. 1

Chemical structure of prednisolone.


Fig. 2 Second derivative spectra of prednisolone standard:

maximum, 241 nm.

Spectrophotometric analyses
Apparatus. Spectrophotometric analyses were performed with a
microcomputer-based Systronics Lambda-6 UV-Visible double
beam spectrophotometer equipped with a Wep 800DX printer.
It was interfaced with a Wep 800DX Data Station via a standard
RS232C interface for storage of spectra. The Wep printer
(Model 800DX) was linked to the data station. Suitable settings
are: slit width 2 nm, response time 5 s, scan speed 60 nm/min
and second derivative mode. A 10-mm quartz cuvette suitable
for the far-UV region was used in this study.
Reagents and solutions. Prednisolone, bulk drug was kindly
made available from Mahima Exports, Sonepat (India).
Prednisolone tablets were purchased from the market. All other
reagents were of analytical reagent grade.
Analysis of tablet. Preparation of prednisolone standard
solutions and calibration: A stock solution containing 200 g/ml
of prednisolone was prepared by dissolving 0.020 g of
prednisolone in methanol, then transferring the mixture into a
100 ml calibrated flask and diluting the volume up to the mark
with distilled water. Prednisolone solutions containing 20
g/ml were tested for stability in solution and during the actual
analysis. The behavior of the analyte remained unchanged up to
about 1 month from their preparation. Further tests of stability
(i.e., beyond 1 month) were found unnecessary and were not
made. All measurements were made at room temperature. The
standard solutions were prepared by the proper dilution of the
stock standard solution with doubly distilled water to reach the
concentration range of 2 50 g/ml.
HPLC method
Apparatus. The HPLC system consisted of a Waters (Milford,
MA, USA) analytical liquid chromatograph equipped with
reversed-phase 300 3.9 mm i.d. 10 m particles, a Bondapak
ODS (C18) column, a 510 HPLC pump, a 717 Plus autosampler,
variable-wavelength 480 UV detector and a 746 data module,
all from Waters. The column and the HPLC system were kept
in ambient conditions. The mobile phase was acetonitrile
citrophosphate buffer (pH 5; 45:55 v/v). This was filtered through
a 0.22-m membrane filter using a Millipore HPLC solvent
filtration assembly, and was delivered at a flow rate of 1.2 ml/min.
The injection volume was 20 l. The elute was analyzed at a
wavelength of 241 nm with detector range setting fixed at 0.01
Reagents and solutions.
Prednisolone and 11-hydroxyprogesterone, both USP reference standards were obtained
commercially from Sigma (Stockholm, Sweden). Methanol was
of HPLC grade; phosphoric acid, citric acid and disodium


Fig. 3 Second derivative spectra of prednisolone tablet (200 400

nm): maximum, 250 nm.

hydrogen phosphate were of analytical reagent grade, all from

Merck (India). Highly pure water, as prepared by a Millipore
purification system, was used for preparation of all aqueous
standard and buffer solutions. Prednisolone formulations were
obtained from local market. Stock solutions of prednisolone (20
mg/ml) and 11-hydroxyprogesterone (internal standard (IS) 10
mg/ml) were prepared in methanol. Appropriate dilutions of
these solutions were made with distilled water to produce
working solutions containing 0 300 g/ml for prednisolone and
100 g/ml for internal standard. Calibration standards containing
0 300 g/ml prednisolone and 100 g/ml of internal standard
were prepared by diluting the working solution with distilled
water. The solutions were prepared as needed. The prepared
dilutions were injected serially. The obtained peaks were
integrated and the area under the curve (AUC) was calculated.
The stability of the solution of prednisolone during analyses was
determined by repeated analyses of samples during the course of
the experiment on the same day and also on different days after
storing at laboratory bench conditions and in the refrigerator.
Procedure to prepare the citrophosphate buffer (pH 5.0): Mix
48.5 ml of 0.1 M citric acid with sufficient 0.2 M disodium
hydrogen phosphate to produce 100 ml. Solution of citric acid
for citrophosphate buffer (pH 5.0): 3.6 g of citric acid in 110 ml
of water. Solution of disodium hydrogen phosphate for
citrophosphate buffer (pH 5.0): 10.74 g of disodium hydrogen
phosphate in 150 ml of water.
Analysis of tablets. Commercially available formulations of
prednisolone were estimated by the proposed method. For each
brand, three samples were thoroughly mixed and an accurately
measured aliquot amount (equivalent to 5 mg of prednisolone)
was transferred to a series of 25 ml volumetric flasks (five in
each case); the volume was adjusted by methanol and the contents
were analyzed. From the AUC, the drug content was calculated.

Results and Discussion

In this study, quantitative determinations of prednisolone in
tablets were performed by second-order derivative UV
spectrophotometric method and HPLC method.
derivative UV spectrophotometry eliminates the possible
scattering effects of colloidal particles and the turbidity
problems, it was decided to investigate this method for routine
analysis. It is simple, rapid and sensitive and also it does not
require any pretreatment procedure. Second-order derivative
spectra of prednisolone standard and tablet are shown in Figs. 2
and 3, respectively. Regression analysis for the second-order
derivative UV spectrophotometric method was carried out
(Table 1) and the linearity of the calibration graph and
adherence of the method to Beers law were validated by the
high value of the correlation coefficient (r), 0.9999.



Table 1 Statistical analysis for the calibration curve of

prednisolone in tablets
Analytical Linearity
wavelength/ range/
g ml1
(n = 5)


2 50


0 300


Y = 0.01317C
Y = 258439C +


a. C is concentration of analyte/g ml1.

Table 2 Results obtained in the determination of prednisolone
in tablets
Amount labeled/
mg tablet1

Amount founda/
mg tablet1











(n = 5)

Fig. 4 Typical RP-HPLC chromatogram of prednisolone spiked

with internal standard.

Table 4 Comparison of methods for the determination of

prednisolone in tablets, ANOVA test

a. Mean of three experiments.

Table 3

Recovery analysis of prednisolone in tablets

mg tablet1

(n = 5)











a. Mean of three experiments.

Quantitative determination of prednisolone in tablets using

derivative UV spectrophotometric method was performed and
the results were in good agreement with the labeled amount of
prednisolone (Table 2). In addition, coefficient of variation
(CV) for the determination of prednisolone was 1.38. Closeness
of the amount found to the labeled amount and the low
coefficient of variation value showed that the proposed method
was accurate and precise. A recovery study conducted by the
derivative UV spectrophotometric method was performed by
spiking the powdered tablets with appropriate amounts of stock
solution. The results of the recovery analyses are also
represented in Table 3. High recovery, 96.2% and low standard
deviation confirmed the suitability of the proposed method for
the determination of prednisolone in different pharmaceutical
Determination of prednisolone was also
conducted by a modified isocratic reversed-phase highperformance liquid chromatographic method.
Figure 4
represents a typical chromatogram obtained for the analysis of
standard prednisolone. As shown in this figure, the standard
prednisolone solution was eluted, forming a well-shaped peak,
and was well separated from the solvent front. Therefore, no
additional extractions or separations were required. The high
correlation coefficient value (Table 1) and low standard
deviation (Table 2) proved that HPLC method was precise and
accurate. In addition, a relatively high recovery value (98.0%,
Table 3), was obtained. Furthermore, the results obtained with
HPLC were in good agreement with those obtained by second
order derivative UV spectrophotometric method. Lastly, results
obtained by these methods for the determination of prednisolone
in tablets were compared by analysis of variance (ANOVA) test

Amount labeled
(10 mg/tablet)



Amount founda



a. Average of five experiments.

ANOVA, P = 0.05; Fcalculated = 0.762; Ftheoretical = 3.89.

(Table 4) and there was no significant difference (P = 0.05)

between the results. The derivative spectrophotometric method
was simple, rapid and accurate. Such features render it suitable
for the routine analysis in quality control laboratories. The
proposed derivative UV spectrophotometric method can also be
applied for the determination of prednisolone present in a
mixture together with another active substance that absorbs at
the same wavelength range as the prednisolone or metabolites
of prednisolone. We conclude that the sensitivities of proposed
second-order derivative UV spectrophotometric method and
HPLC method are almost comparable and both the methods are
suitable for the analysis of prednisolone in commercial tablets.

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