Professional Documents
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23
2007 The Japan Society for Analytical Chemistry
1241
Notes
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)
Introduction
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.
E-mail: dhruvks123@rediffmail.com
Fig. 1
1242
Experimental
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
AUFS.
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
1243
250
2 50
241
0 300
Correlation
coefficient,
r
Regression
equationa
Y = 0.01317C
0.0366
Y = 258439C +
39846
0.9999
0.9965
Amount founda/
mg tablet1
SD
CV
10
9.8
0.48
1.38
10
10.1
0.69
2.04
Second-order
(n = 5)
HPLC
Second-order
(n = 5)
HPLC
10
0.5
0.481
96.2
0.07
10
0.5
0.490
98.0
0.12
Amount labeled
(10 mg/tablet)
Second-order
HPLC
Amount founda
CV
9.8
1.38
10.1
2.04
References
1. S. Gorog, Anal. Sci., 2004, 19, 767.
2. L. Novakova, P. Solich, and L. Matysova, Anal. Bioanal.
Chem., 2004, 379, 781.
3. S. Gorog, J. Pharm. Biomed. Anal., 2005, 36, 931.
4. S. Gorog, Fresenius Z. Anal. Chem., 1981, 309, 97.
5. S. Gorog, Fresenius Z. Anal. Chem., 1998, 362, 4.
6. M. Blanco, J. Coello, H. Iturriaga, S. Maspoch, and N.
Villegas, Analyst, 1999, 124, 911.
7. J. Lindholm, D. Westerlund, K. Karlsson, K. Caldwell, and
T. Fornstedt, J. Chromatogr., A, 2003, 992, 85.
8. E. J. Kikta and J. Stange, J. Chromatogr., 1977, 138, 41.
9. The United States Pharmacoepia 26, 2003, United States
Pharmacoepial Convention, Rockville, MD, 2149.
10. British Pharmacopoeia, 2000, Her Majestys Stationery
Office, London, 1335.
11. J. Lindholm, M. Johansson, and T. Fornstedt, J.
Chromatogr., B, 2003, 791, 323.
12. R. E. Graham, E. R. Biehl, and M. J. Uribe, J. Assoc. Off.
Anal. Chem., 1983, 66, 264.