American Journal of Anal yt ical Chemistry, 2010, 1, 144-149
doi:10.4236/ajac.2010.13018 Published Online November 2010 (http://www.SciRP.org/journal/ajac)
Validated HPTLC Method for Simultaneous Estimation of
Isotretinoin and Erythromycin in Bulk Drug and Topical
Gel Form
Atul S. Rathore, Lohidasan Sathiyanarayanan, Kakasaheb R. Mahadik*
Department of Ph arm ace ut i ca l Chemist ry, Poona College of Pharmacy,
Bharati Vidyapeeth University, Pune, India
Received June 16, 201 0; revised August 2, 2010; accepted October 26, 2010
Abstract
A simple, precise and accurate high performance thin layer chromatographic method has been developed for
the simultaneous estimation of Isotretinoin and Erythromycin in pharmaceutical gel. The separation was car-
ried out on Merck TLC aluminum sheets of silica gel 60 F254, (20 × 10 cm) with 250 µm thickness using
toluene: DMSO: methanol (6.5:0.2:2.5, v/v/v) as a mobile phase. HPTLC separation of the two drugs fol-
lowed by densitometric measurement of their spots at 340 nm for Isotretinoin before derivatization and 410
nm for Erythromycin after derivatization with 10% H2SO4 and heating at 100C for 15 min. The drugs were
satisfactorily resolved with RF values of 0.38 ± 0.02 and 0.55 ± 0.02 for Isotretinoin and Erythromycin, re-
spectively. The accuracy and reliability of the method was assessed by evaluation of linearity (30-150 ng
spot-1 for Isotretinoin and 1200-6000 ng spot-1 for Erythromycin), precision (intra-day RSD 0.62-0.79% and
inter-day RSD 0.43-0.71% for Isotretinoin and intra-day RSD 0.47-1.71% and inter-day RSD 0.42-1.49% for
Erythromycin), accuracy (98.91 ± 0.92% for Isotretinoin and 99.27 ± 0.72% for Erythromycin), and specific-
ity, in accordance with ICH guidelines.
Keywords: Isotretinoin, Erythromycin, HPTLC, Validation
1. Introduction
Isotretinoin (Figure 1(a)) is chemically (13cis)-retinoic
acid. Isotretinoin is a member of the large group of Vi-
tamin A related compounds. It alters DNA transcription
and decreases the size and sebum output of the sebaceous
glands. It also stabilizes keratinization, Due to its effect
on regulating cell differentiation it has been used for the
treatment of cystic and nodular acne and also as an in-
hibitor of neoplasic cells proliferation [1-7]. Erythromy-
cin (Figure 1(b)) is chemically (3R, 4S, 5S, 6R, 7R, 9R,
11R, 12R, 13S, 14R)-6-{[(2S, 3R, 4S, 6R)-4-(di- me-
thylamino)-3-hydroxy-6-methyloxan-2-yl]oxy}-14-
ethyl-7, 12, 13-trihydroxy-4-{[(2R, 4R, 5S, 6S)-5-hy-
droxy-4-methoxy-4,6-dimethyloxan-2-yl]oxy}-3, 5, 7, 9,
11, 13-hexamethyl-1-oxacyclotetradecane-2,10-dione. It
is an antibiotic that possess bactericidal activity, particu-
larly at higher concentrations. It acts by binding to the
50s subunit of the bacterial 70s rRNA complex, protein
synthesis and subsequently structure/function processes
critical for life or replication are inhibited [8,9]. Litera-
ture review reveals that several analytical methods have
been reported for Isotretinoin [10-12] and Erythromycin
[13-18] as individual determination or in biological fluids
or in combination with other drugs in pharmaceutical
dosage forms. A comprehensive literature survey re-
vealed that no method has been reported for simultane-
ous estimation of Isotretinoin and Erythromycin by
HPTLC in pharmaceutical dosage forms. So, the present
study is designed for the development and validation of
simple HPTLC method for the simultaneous estimation
of Isotretinoin and Erythromycin in their combined topi-
cal gel formulation. The proposed method is validated as
per ICH guideline [19].
2. Experimental
2.1. Materials
Working standards of pharmaceutical grade Isotretinoin

A. S. RATHORE ET AL.
145
(a)
(b)
Figure 1. (a) Structure of Isotretinoin; (b) Structure of
Erythromycin.
and Erythromycin were obtained as a gift sample from
Ranbaxy laboratory ltd. Dewas, India. Formulation in the
form of gel (Isotrexin) was procured as a gift sample
from Stiefel Laboratories (Ireland) Ltd., Silgo, Ireland
containing 0.05% Isotretinoin and 2% Erythromycin
(w/w). All chemicals and reagents of analytical grade
were purchased from Merck Chemicals, Mumbai, India.
High purity deionized water was obtained from Millipore,
Milli-Q (Bedford, MA, USA) wate r p ur ification system.
2.2. Preparation of Standard Stock and Working
Solutions
Stock standard solution containing Isotretinoin (150 µg
mL-1) and Er yth ro mycin (6 000 µg mL-1) w as pr ep ar ed by
dissolving 7.5 mg Isotretinoin and 300 mg Erythromycin
in methanol in a 50 mL volumetric flask. Working stan-
dard solution of Isotretinoin and Erythromycin was pre-
pared at concentration of 15 ng µL-1 and 600 ng µL-1
respectively, by diluting the stock standard solution in
methanol. The stock solution was stored at 2-8°C pro-
tected from light.
2.3. Instrumentation
The samples were spotted in the form of bands 6 mm
width with a Camag 100 microlitre sample syringe
(Hamilton, Bonaduz, Switzerland) on silica gel precoated
aluminum plate 60 F254, [(20 × 10 cm) with 250 µm
thickness; E. Merck, Darmstadt, Germany, supplied by
Anchrom Technologists, Mumbai] using a Camag Lino-
mat IV applicator (Switzerland). The plates were pre-
washed with methanol and activated at 110°C for 5 min
prior to chromatography. A constant application rate of
0.1 µLs-1 was used and the space between two bands was
6 mm. The slit dimension was kept at 5 mm × 0.45 mm
and the scanning speed was 10 mm s-1. The mobile phase
was consisted of toluene: DMSO: methanol (6.5:0.2:2.5,
v/v/v) and 18.4 mL were used per chromatography run.
Linear ascending development was carried out in 20 cm ×
10 cm twin trough glass chamber (Camag, Muttenz,
Switzerland) saturated with the mobile phase. The opti-
mized chamber saturation time for mobile phase was 30
min at room temperature (25 ± 2°C). The length of chro-
matogram run was 8 cm. Densitometric scanning was
performed using a Camag TLC scanner III in the
reflectance-absorbance mode and operated by CATS
software (V 3.15, Camag). The sources of radiation used
were deuterium and tungsten lamp with a spectral range
from 190 to 800 nm. Concentrations of the compound
chromatographed were determined from the intensity of
the diffused light. Evaluation was by peak areas with
linear regression.
2.4. Selection of Analytical Wavelength
A UV spectrum for the solution (10 µg mL–1) of Isot-
retinoin was recorded in a 10 mm cell over the range
200-400 nm using methanol in the reference cell. Isot-
retinoin showed maximum absorbance at 340 nm (Fig-
ure 2) while Erythromycin was detected at 410 nm after
derivatization with 10% H2SO4 and heating at 100 °C for
15 min [13].
2.5. Optimization of HPTLC Method
The HPTLC procedure was optimized with a view to
develop a simultaneous assay method for Isotretinoin and
Erythromycin. The stock standard solution containing
Figure 2. UV spectrum of Isotretinoin.

146 A. S. RATHORE ET AL.
150 µg mL-1 of Isotretinoin and 6000 µg mL-1 of Eryth-
romycin were spotted onto Merck HPTLC silica gel pre-
coated aluminum plate 60 F254, (20 × 10 cm) with 250
µm thickness and run in different solvent systems. Ini-
tially, toluene, ethyl acetate and methanol were tried in
different ratio. (Toluene, acetone and methanol) and
(hexane, acetone and methanol) were tried in various
ratio. Finally, toluene, DMSO and methanol were tried.
It was found that the DMSO is responsib le for th e elu tion
of erythromycin. The optimum mobile phase was found
to be consisted of toluene: DMSO: methanol (6.5:0.2:2.5 ,
v/v/v). The drugs were satisfactorily resolved with RF
values at 0.38 ± 0.02 and 0.55 ± 0.02 for Isotretinoin and
Erythromycin, respectively. In order to reduce the neck-
less effect the TLC chamber was saturated for 30 min
using saturation pads. The mobile phase was run up to a
distance of 8 cm; which takes approximately 45 min. for
complete development of the TLC plate.
2.6. Validation of HPTLC Method
The optimized HPTLC method was validated with re-
spect to the following Parameters. The validation was
performed as per the ICH guideline [19].
2.6.1. Linearity
Stock standard solution containing 150 µg mL-1 of Isot-
retinoin and 6000 µg mL-1 of erythromycin was further
diluted with methanol to obtain a working standard solu-
tion at concentration of 15 ng µL-1 and 600 ng µL-1 re-
spectively. From the working standard solution 2, 4, 6, 8,
10 µL volumes were spotted on HPTLC plate to obtain a
final concentration range of 30-150 ng spot-1 for Isotreti-
noin and 1200-6000 ng spot-1 for Erythromycin. Each
concentration was applied six times on the TLC plate.
The plate was then developed using the previously de-
scribed mobile phase. Curves were obtained by plotting
the peak area against concentration of the drug. Linear
calibration curves were generated using least-squares
linear-regression analysis.
2.6.2. Precision
The precision of the method was verified by repeatab ility
(intraday) and intermediate precision studies. Repeatabil-
ity studies were performed by analysis of three different
concentrations of 30, 90, 150 ng spot-1 and 1200, 3600,
6000 ng spot-1 for Isotretinoin and Erythromycin, respec-
tively. Method repeatability was achieved from RSD %
values obtained by repeating the assay six times on the
same day for intra-day precision. The intermediate (in-
terday) precision of the method was checked by per-
forming same procedure on different days under the
same experimental conditions.
2.6.3. Robus tness
The robustness was studied by evaluating the effect of
small but deliberate variations in the chromatographic
conditions. Following the introduction of small changes
in the mobile phase composition (± 0.1 mL for each
component), the effects on the results was examined.
Mobile phases having different compositions, e.g. tolu-
ene: DMSO: methanol (6.5:0.2:2.5, v/v/v), (6.5:0.2:2.4,
v/v/v), (6.5:0.2:2.6, v/v/v), were tried and chroma-
tograms were run. The amount of mobile phase was var-
ied over the range of ±5%. The time from spotting to
chromatography and from chromatography to scanning
was varied from ±10 min. The robustness of the method
was determined at three different concentration levels of
30, 90, 150 ng spot-1 for Isotretinoin and 1200, 3600,
6000 ng spot-1 fo r E ry thromycin.
2.6.4. Specifi c ity
The ability of an analytical method to unequivocally as-
sess the analyte in the presence of other components can
be demonstrated by evaluating specificity. The specificity
of the HPTLC method was determined by analyzing
standard drug and test samples. The spot for Isotretinoin
and Erythromycin in the samples was confirmed by
comparing the RF and spectrum of the spot to that of a
standard. The peak purity of Isotretinoin and Erythro-
mycin was determined by comparing the spectrum at
three different regions of the spot i.e. peak start (S), peak
apex (M) and peak end (E).
2.6.5. Accuracy
Accuracy of the HPTLC method was carried out by ap-
plying the method to drug sample [Isotretinoin 0.05%
and Erythromycin 2% (w/w) combination in gel form of
alcoholic base of 30 g tube] to which known amount of
Isotretinoin and erythromycin standard powder corre-
sponding to 50, 100 and 150% of label claim was added
(standard addition method). The absolute recovery was
calculated by comparing the peak areas obtained from
standard solution of Isotretinoin and Erythromycin with
the peak areas of samples of different concentration.
2.6.6. Anal ysis of a Marketed Formul a ti o n (Assay)
Isotrexin Gel 30 g tube (labeled to contain Isotretinoin
0.05% and Erythromycin 2% (w/w) in gel form of alco-
holic base of 30 g tube, Stiefel India Ltd.) means 15
mg/30 g of Isotretinoin and 600 mg/30 g of Erythromy-
cin. An accurate weight of the 1 gram of gel equivalent
to 0.5 mg of Isotretinoin and 20 mg of Erythromycin was
transferred into a 10 mL volumetric flask containing 8
mL methanol and sonicated for 30 min. The contents
were restored to room temperature and diluted to volume
with methanol to furnish stock test solution. The stock

A. S. RATHORE ET AL.
147
solution was filtered through a 0.45 µm Nylon syringe
filter. From the stock test solution (contain ing 50 ng µL-1
of Isotretinoin and 2000 ng µL-1 of Erythromycin), 2 µL
volume was spotted for six times to achieve a final con-
centration of 100 ng spot-1 and 4000 ng spot-1 for Isot-
retinoin and Erythromycin, respectively.
3. Results and Discussion
In this work HPTLC method for the analysis of Isotreti-
noin and Erythromycin in topical gel form was devel-
oped and validated as per ICH, Q2 (R1), guideline.
3.1. Optimization of Procedures
The experimental conditions for HPTLC such as wave-
length of detection and mobile phase composition were
optimized to provide accurate, precise and reproducible
results for the simultaneous determination of Isotretino in
and Erythro mycin. A scanning w aveleng th of 340 n m for
Isotretinoin was obtained from UV spectrum and 410 nm
for Erythromycin after derivatization with 10% H2SO4
and heating at 100°C for 15 min [13]. A good resolution
was obtained by using an optimum mobile phase con-
sisted of toluene: DMSO: methanol (6.5:0.2:2.5, v/v/v).
Isotretinoin and Erythromycin were satisfactorily re-
solved with RF values at 0.38 ± 0.02 and 0.55 ± 0.02,
respectively (Figure 3).
3.2. Method Validation
3.2.1. Linearity
Linear relationships were observed by plotting drug
concentrations against peak areas for each compound.
Isotretinoin and Erythromycin showed linear response in
the concentration range of 30-150 ng spot-1 and 1200-
6000 ng spot-1, respectively. The corresponding linear
regression equation was y = 3.774x + 468.7 and y = 2.22x
– 974.1 with square of correlation coefficient (R2) of
0.997 ± 0.066 and 0.996 ± 0.071 for Isotretinoin and
Erythromycin, respectively (Table 1).
3.2.2. Precision
The results of the repeatability and intermediate preci-
sion experiments are shown in Table 2. The developed
methods were found to be precise as the RSD values for
repeatability and intermediate precision studies were <
2%, respectively as recommended by ICH guidelines.
3.2.3. Robustness
The standard deviation of the peak areas was calculated
for each parameter and the RSD was found to be less
than 2% for HPTLC. The low values of the RSD%, as
shown in Table 3. indicated the robustness of the pro-
posed method.
3.2.4. Specifi c ity
The peak purity of Isotretinoin and Erythromycin was
assessed by comparing their respective spectra at the
peak start, apex and peak end positions of the spot i.e., r
(S, M) = 0.9979 and r (M, E) = 0.9986. A good correla-
tion (r = 0.9981) was also obtained between the standard
and sample spectra of Isotretinoin and Erythromycin.
Table 1. Linear regression data for the calibration curves.
Parameters Isotretinoin Erythromycin
Linearity rang e 30-150 ng spot-1 1200-6000 ng spot-1
r2 ± S.D. 0.997 ± 0.066 0.996 ± 0.071
Slope ± S.D. 3.774 ± 0.042 2.22 ± 0.099
Intercept ± S.D 468.7 ± 0.528 974.1 ± 0.984
(a)
(b)
Figure 3. (a) Densitogram of Isotretinoin 60 ng spot-1 before
derivatization at 340 nm; (b) Densitogram of Erythromycin
400 ng spot-1 after derivatization at 410 nm. 2

A. S. RATHORE ET AL.
148
Table 2. Precision studies of Isotretinoin and Ery thromycin.
Repeatability (n = 6) Intermediate precision (n = 6)
Drugs Conc. (ng spot-1) Found conc. ± SD RSD (%) Found conc. ± SD RSD (%)
30 29.53 ± 0.18 0.62 29.97 ± 0.13 0.43
90 90.08 ± 0.09 0.10 90.86 ± 0.64 0.71
Isotretinoin
150 152.88 ± 1.21 0.79 152.14 ± 0.69 0.45
1200 1067.00 ± 15.47 1.45 1038.89 ± 4.40 0.42
3600 3690.23 ± 17.44 0.47 3637.13 ± 54.99 1.49
Erythromycin
6000 5993.19 ± 101.37 1.71 5934.68 ± 59.99 1.02
Table 3. Robustness testing for HPTLC method.
SD of peak area % RSD
Parameter Isotretinoin Erythromycin Isotretinoin Erythromycin
Mobile phase composition (± 0 .1 ml) 1.54 1.53 1.24 1.09
Amount of mobile phase (± 5 %) 1.82 0.65 1.34 1.27
Time from spotting to ch romatography ( ± 10 min) 0.70 0.94 0.40 0.89
Time from chromatography to scanning (± 10 min) 0.72 0.59 0.48 0.40
Table 4. Accuracy of the proposed method.
For HPTLC (n = 6)
Drugs Label claim
mg g-1 of gel Amount added in mg (%) Total amount (mg) Actual conc. taken (ng spot-1)calculated conc. ± SD RSD (%) Recovery (%)
0.25 (50%) 0.75 75 73.69 ± 0.38 0.52 98.26
0.5 (100%) 1.00 100 98.75 ± 1.29 1.31 98.75 Isotretinoin 0.5
0.75 (150%) 1.25 125 124.65 ± 1.14 0.92 99.72
10 (50%) 30 3000 2942.10 ± 17.36 0.59 98.07
20 (100%) 40 4000 4062.00 ± 17.22 0.42 101.55
Erythromycin 20
30 (150%) 50 5000 4909.50 ± 56.37 1.15 98.19
3.2.5. Accuracy
As shown from the data in Table 4, satisfactory recover-
ies % with small relative standard deviations, RSD (%)
were obtained at various added concentrations for both
the drugs. The results indicate the methods are highly
accurate for simultaneous determination of the two
drugs.
3.2.6. Anal ysis of a Marketed Formul a ti o n (Assay)
Using the proposed chromatographic method, assay of
Isotretinoin and Erythromycin in their formulation in gel
form was carried out. Satisfactory results were obtained
for both drugs in a good agreement with the lab el claims.
The recovery % ± RSD % of six replicate determinations
were 98.78 ± 0.99 (Isotretinoin), 101.19 ± 1.05 (Eryth-
romycin) by HPTLC (Table 5).
4. Conclusions
The developed TLC technique is precise, specific and

A. S. RATHORE ET AL.
149
Table 5. Analysis of marketed formulation (assay) of isot-
retinoin and erythromycin.
For HPTLC (n = 6)
Drugs Label claim %
in 30 g gel Drug content
(%) ± SD RSD (%)
Isotretinoin 0.05 % 98.78 ± 0.98 0.99
Erythromycin 2 % 101.19 ± 1.06 1.05
accurate. Statistical analysis proves that the method is
suitable for analsizing of Isotretinoin and Erythromycin
as bulk drug and in pharmaceutical gel formulation. The
proposed TLC method is less expensive, simpler, rapid,
and more flexible than LC.
5. Acknowledgements
The authors would like to thank, Ranbax y laboratory ltd.
Dewas, India for the gift samples and Mr. Vijayanand
Chakrapani QA-Manager Stiefel India Pvt. Ltd. for ar-
ranging the gift sample of formulation (Isotrexin gel) and
Poona College of Pharmacy, Bharati Vidyapeeth Univer-
sity for providing facilities and encouragement for car-
rying out this study.
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