Validated HPTLC Method for Assay of Prednisolone

in Tablets and Comparison with Pharmacopeial Methods

Astha Mehta* and Anil Thaker

Key Words
HPTLC
Prednisolone
Tablets
Validation

Summary
A rapid and reliable high-performance thin-layer chromatographic
method has been established for analysis of prednisolone in a tablet
dosage form. The prednisolone standard solution and sample were
applied to precoated silica gel G60 F254 HPTLC plates, prewashed
with methanol, and the plate was developed with 95:5 (v/v) chloro-
form–methanol as mobile phase. Quantification was by densitome-
try at 250 nm. A calibration plot was established showing the depen-
dence of response (peak area) on the amount chromatographed in
the range 2–10 μg per band (r = 0.9967). The method was quantita- Figure 1
tively validated for specificity, accuracy, precision, repeatability, The structure of prednisolone.
and robustness to prove its suitability for analysis of the tablet
dosage form. The method was also compared with pharmacopeial
methods for assay of prednisolone and the results confirmed statis-
tically that the method can be used as a substitute for the pharma-
copeial methods. HPLC [2] and UV [3] methods have been reported in the phar-
macopoeia and the literature contains other methods for analysis
of prednisolone in tablets and biological fluid [4–7].

1 Introduction
Prednisolone, (11β)-11,17,21-trihydroxypregna-1,4-diene-3,20-
dione, Figure 1, an anti-allergic, anti-inflammatory glucocorti-
coid drug [1] is widely used to treat in rheumatoid arthritis and
is generally well tolerated. The principle behind its action in the
body is its negative feedback mechanism to the hypothalamus.
Prednisolone stimulates the adrenal cortex to release glucocorti-
coid. The drug mimics the function of endogenous glucocorti-
coids by inhibiting edema, fibrin deposition, capillary dilation,
leukocyte migration, capillary proliferation, deposition of colla-
gen, and scar formation associated with inflammation caused by
pathogens, or chemical or physical stimuli. The active ingredi-
ent prednisolone and its tablets are official in USP, BP, and IP.
A. Mehta, Department of Pharmaceutical Chemistry, School of Pharmacy and
Technology Management, NMIMS University, Vile Parle-W Mumbai-400056,
India; and A. Thaker, Department of Quality Assurance, School of Pharmacy and
Technology Management, NMIMS University, Vile Parle-W, Mumbai-400056,
India.
E-mail: astha2212@gmail.com
A few TLC methods have been reported for qualitative analysis
of prednisolone and, mainly, other corticosteroids in biological
fluids [8–10]. The TLC methods reported do not enable quanti-
tative analysis of the drug, and the methods used for extraction
of biological samples are complex, as also is mobile-phase
preparation. These methods were used as the basis for develop-
ment of a high-performance thin-layer chromatographic
(HPTLC) method for analysis of prednisolone in its pharmaceu-
tical formulation. Hydrocortisone is related to prednisolone and
has partial mineralocorticoid action leading to side effects, for
example salt retention; its level is therefore critical and is con-
trolled up to 1%. The wavelength of maximum absorbance of
hydrocortisone is 240 nm, which is very close to that of pred-
nisolone, making it very difficult to detect its presence in a pred-
nisolone formulation by use of a UV method. Estimation of the
level of hydrocortisone in prednisolone tablets by HPLC is too
time consuming and intricate for implementation of the method
to a large number of batches. Thus for assay of a formulation it
is important to separate the two and to overcome the drawbacks
of other methods reported for assay.
In this paper we report the development and validation of a
rapid, simple, specific, sensitive, accurate, and precise HPTLC
method for analysis of prednisolone in the tablet dosage form.

208 Journal of Planar Chromatography 23 (2010) 3, 208–211

0933-4173/$ 20.00 © Akadémiai Kiadó, Budapest
DOI: 10.1556/JPC.23.2010.3.8

Figure 2
Typical densitogram obtained from prednisolone standard.
Figure 3
Typical densitogram obtained from prednisolone standard spiked
with related substance hydrocortisone.
2 Experimental
2.1 Chemicals, Materials, and Solutions
Prednisolone and hydrocortisone working standard, pred-
nisolone tablet placebo, and prednisolone tablets (5 mg BP; LPC
Medical, UK) were generous gifts from Rusan Pharmaceutical,
Mumbai, India. Chloroform of AR grade and methanol of HPLC
grade were from Merck, Mumbai, India.
Working standard of prednisolone (25 mg) was accurately
weighed, transferred to a 25-mL volumetric flask, dissolved in
methanol, and diluted to volume to give a stock solution of
1 mg mL–1. This stock solution (5 mL) was further diluted to
10 mL with methanol to give a working standard of 0.5 μg μL–1.
This solution (10 μL) was applied to the plates to furnish 5 μg
per band.
2.2 Sample Preparation
Twenty tablets were crushed and ground to a fine powder.
A mass of powder equivalent to 5 mg prednisolone was trans-
ferred to a 10-mL volumetric flask and the drug was extracted
with 5–8 mL methanol with the aid of sonication for 15 min. The
final volume was diluted to 10 mL with methanol to give sample
solution containing 0.5 μg μL–1 prednisolone. Working standard
solution of concentration 0.5 μg μL–1 and the sample solution
(10 μL of each) were analyzed by HPTLC.
2.3 Chromatography
HPTLC was performed on 20 cm × 20 cm aluminum foil-
backed plates coated with 0.2 mm layers of silica gel 60 F254
Journal of Planar Chromatography 23 (2010) 3
Assay of Prednisolone in Tablets
(E. Merck, Darmstadt, Germany). Before use plates were pre-
washed with methanol. Samples were applied to the plates as
5-mm bands, 5 mm apart, by means of a Desaga (Wiesloch,
Germany) AS 30 sample applicator; the spraying rate was
7 s μL–1. Plates were developed to a distance of 7.5 cm from
the lower edge of the plate, with 95:5 (v/v) chloroform–
methanol as mobile phase, in a twin-trough developing cham-
ber previously saturated with mobile phase vapor for 30 min.
After development the plates were dried and then scanned at
250 nm, the wavelength of maximum absorption of pred-
nisolone and at which the impurity hydrocortisone was also
detected, by means of a Desaga CD 60 densitometer with Pro-
quant software (version 1.3 ) and zoom browser software. The
slit dimensions were 4.0 mm × 1.0 mm. Peak areas were used
for calculation.
2.4 Method Validation
The method was validated for linearity, specificity, accuracy,
intra-day and inter-day precision, reproducibility of sample
application, robustness, and stability in solution. The limits of
quantification and of detection for prednisolone were also deter-
mined.
To measure linearity of detector response prednisolone solution
of concentration 1 μg μL–1 was prepared and used to apply
2–10 μg per band to the plate (by varying the volume applied).
Photometric measurements were performed and the peak area of
each band was recorded. The specificity of the method was
checked by chromatography of working standard, placebo,
related substance (hydrocortisone), and sample solution of pred-
nisolone extracted from tablets. The accuracy of the method was
evaluated by measurement of recovery. A known amount of drug
standard was added to the tablet placebo at three different levels,
80, 100, and 120% of the amount present in the tablet. The sam-
ples were extracted, the extract chromatographed, and average
recovery was calculated. Intra-day precision was determined by
analyzing prednisolone standard and six sample solutions of
concentration 5 μg per band on the same day. Inter-day precision
was determined by analyzing standard and six sample solutions
on the next day. The reproducibility of sample application was
assessed by application 10 μL prednisolone standard solution to
a plate, developing the plate, scanning the prednisolone spots,
and calculating the relative standard deviation (RSD, %) for
measurement of peak area. The robustness of the method was
checked by spotting standard and sample solution on the plate
and developing the plate after slightly altering the conditions.
The conditions changed were the ratio of chloroform to
methanol in the mobile phase (from 95:5 to 97:3 and 93:7), the
band width (from 5 mm to 6 or 7 mm), and the detection wave-
length (from 250 nm to 260 or 240 nm). Solution stability was
tested by analyzing the sample solution initially and then the
same solution after 8 h, by comparison with freshly prepared
standard solution. The limits of detection and limit of quantifi-
cation were estimated by visual inspection and on the basis of
the standard deviation of the response and the slope of the cali-
bration plot.
2.5 Comparison with Pharmacopeial Methods
When developing a new analytical method, it is desirable to
compare the results from the new method with those from an
accepted method. Statistical methods can be used to check
209
Assay of Prednisolone in Tablets

Table 1 Table 3

Analysis of prednisolone in the tablet dosage form. Method validation data.

Label claim [mg] 5 Linear range [μg per band] 2–10

Amount found ± SDa) [mg] 4.92 ± 0.12 Correlation coefficient 0.9969
Assay ± SDa) [%] 98.61 ± 2.1 Limit of detection 0.2 μg per spot
CV [%] 2.14 Limit of quantification 0.6 μg per spot
a)Average value ± standard deviation (n = 10) Accuracy 100.02%
Precision (RSD, [%])
Repeatability 0.74
Table 2
Inter-day (n = 6) 2.6
Accuracy of HPTLC method for prednisolone (label claim 5 mg per
tablet). Intra-day (n = 12) 2.7
Robustness (RSD, [%])
Amount added to placebo [%] 80 100 120
Change in mobile phase (93:07,
Amount recovered [mg]a) 3.9 5.1 6.0
95:5, 97:3 chloroform–methanol) 0.5
Recovery [%] ± SD 98.3 ± 5.2 101.3 ± 6.6 100.5 ± 7.5
Change in band width (± 1 mm) 1.00
RSD [%] 0.31 0.30 0.29
Change in wavelength (± 10 nm) 96–90%
a)Each reading is the mean from triplicate analysis Stability of solution (RSD, [%]) 1.1
Specificity Specific

whether one set of results is significantly different from another.

The F test evaluates the difference between the spread of results
and the t test evaluates the difference between the means [11]. Table 4
Solutions of standard and sample were analyzed by the HPTLC Results obtained from analysis of ten tablets by three methods.
method and by the methods recommended in the BP (HPLC
method) and the IP (UV method). Ten individual tablets were No. Content [%] Content [%] Content [%]
extracted and analyzed in duplicate. by HPTLC by HPLC (BP) by UV (IP)

1 96.22 96.01 96.09

2 100.40 99.21 100.65
3 Results and Discussion
3 99.71 98.72 100.07
Literature survey indicated a variety of methods had been 4 97.42 96.69 97.90
reported for analysis of prednisolone. Most were HPLC or UV 5 96.55 95.92 97.03
methods, which are sophisticated, costly, and time consuming.
6 98.44 99.71 99.01
The TLC and HPTLC methods reported were for identification
but not used for quantitative estimation from formulations. It 7 102.68 101.19 102.49
was therefore decided to establish a method for analysis of pred- 8 100.42 100.37 100.65
nisolone using HPTLC, a versatile, speedy, and cost-effective 9 96.85 95.96 96.50
technique.
10 97.46 100.04 97.63
Because prednisolone is freely soluble in methanol, the tablet
Average 98.62 98.38 98.80
powder was extracted with this solvent. Sonication of the sam-
ple matrix for 15 min helped to extract prednisolone completely. RSD 2.1 2.0 2.1
The mobile phase 95:5 (v/v) chloroform–methanol resulted in
optimum migration of prednisolone (RF 0.53 ± 0.02, Figure 2)
and resolution of the drug from its related substance hydrocorti-
sone without interference from other components of the formu- ficient of 0.9969. The limits of detection and limit of quantifica-
lation matrix (excipients). When excess hydrocortisone was tion were 0.2 μg per band and 0.6 μg per band, respectively.
added to the sample solution to check the specificity of the Average recovery, calculated after use of a suitable dilution fac-
method the chromatogram showed the resolution of the drug tor, was 100.02% (Table 2). Intra-day and inter-day coefficients
peak from the related substance peak was 1.52 (Figure 3). The of variation were 2.7% and 2.6%, respectively. Such low values
amount of prednisolone in the tablet formulation was calculated indicate the method is precise. The RSD for repeatability of
by applying a suitable dilution factor and comparing peak areas measurement of peak area was 0.74%. This was well below the
of standard and sample solutions. Assay of prednisolone in the specification of the scanner, and thus indicates it was function-
tablet formulation, by measurement of peak area, was found to ing correctly. When the robustness of the method checked by
be 98.61 ± 2.1% (Table 1). altering the mobile phase composition and the band width RSD
The response to prednisolone standard was a linear function of of assay results were 0.52 and 1.00%. Changing the wavelength
amount in the range 2 to 10 μg per band, with a correlation coef- by ±10 nm resulted in assay results ranging from 96 to 90%.

210 Journal of Planar Chromatography 23 (2010) 3

 Assay of Prednisolone in Tablets

Table 5

Results from F and t tests.

Method F test t test

F calculated F tabulated (95% confidence) T calculated T tabulated (95% confidence)

HPTLC compared with UV 1.00029 3.18 0.1960 2.086

HPTLC compared with HPLC 1.06680 3.18 0.2508 2.086

Because the robustness results obtained were within the accep-
tance limit of 3% RSD, the method is robust to minor changes in
the method conditions. When solution stability was tested ini-
tially and after 8 h the respective assay results was 101.7% and
100.1%. This result shows the drug is stable in the solution used
for analysis.
It was observed that excipients present in the formulation did not
interfere with the prednisolone peak. The resolution between the
related substance and drug was 1.52 when hydrocortisone was
added to the sample preparation. Different validation data [12]
for the method have been summarized in Table 3.
The method was found to be rapid, simple, specific, sensitive,
precise, accurate and robust. When compared with pharma-
copeial methods recommended for prednisolone tablet, similar
results were obtained for ten tablets (Table 4). The results were
evaluated statistically by us of F and t tests and were found to
pass the criteria that the calculated value should be less than
tabulated value (Table 5). Thus it was ensured the method
could be used as a substitute for the pharmacopeial methods to
obtain results for assay and uniformity of content, and can be
used for routine quality-control analysis of prednisolone from
tablets.
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Ms received: March 19, 2009
Accepted: November 18, 2009

Journal of Planar Chromatography 23 (2010) 3 211