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Densitometric determination of betamethasone dipropionate and salicylic

acid in lotions, and validation of the method

Article  in  JPC - Journal of Planar Chromatography - Modern TLC · December 2003

DOI: 10.1556/JPC.16.2003.6.6

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Lestyo Wulandari Gunawan Indrayanto

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Densitometric Determination of Betamethasone
Dipropionate and Salicylic Acid in Lotions, and Validation
of the Method
Listyo Wulandari and Gunawan Indrayanto*

Key Words:
Betamethasone dipropionate
Densitometry
Lotion
Salicylic acid
TLC
Validation

Summary betamethasone dipropionate in the presence of other corticos-

A simple and rapid densitometric method has been developed for
determination of betamethasone dipropionate and salicylic acid in
lotions. The samples were diluted with 96% ethanol and spotted on
precoated silica gel TLC plates which were then eluted with ethanol
(96%)–toluene–chloroform–glacial acetic acid, 6.0 + 20 + 14 + 0.5
(v/v). Quantitative evaluation was performed by measuring the
absorbance reflectance of the betamethasone dipropionate and sal-
icylic acid spots at λ = 250 and 310 nm, respectively. This densito-
metric TLC method is selective, precise, and accurate and can be
used for routine analysis of lotions in pharmaceutical industry qual-
ity-control laboratories.
teroids. As far as we are aware there has been no report of
the simultaneous TLC determination of salicylic acid and
betamethasone dipropionate. The objective of the work reported
herein was to develop a cheap, rapid, and simple validated TLC
method for determination of betamethasone dipropionate and
salicylic acid in pharmaceutical preparations for pharmaceutical
industry quality-control laboratories.
2 Experimental

2.1 Materials and Reagents

1 Introduction
Pharmaceutical preparations containing betamethasone dipropi-
onate and salicylic acid as active ingredients have recently been
marketed in Indonesia as topical lotions [1]. Official methods
for determination of betamethasone dipropionate are HPLC [2,
3] and spectrophotometry [4] whereas salicylic acid has been
assayed by titration [2, 3, 5–7] and by colorimetric [7] methods.
Kedor-Hackman et al. [8] reported the determination of
betamethasone dipropionate and salicylic acid in topical prepa-
rations by HPLC. TLC methods have been described for analy-
sis of mixtures of salicylic acid and other substances [9]. Datta
and Das [10] published a TLC method for the determination of
L. Wulandari, QC Laboratory, Bernofarm Pharmaceutical Company, Buduran,
Sidoarjo, Surabaya 61252, Indonesia (current address: Department of Chemistry,
Faculty of Mathematics and Natural Sciences, University of Jember, Jember
68131, Indonesia), and G. Indrayanto, Plant Biotechnology Research Group, Fac-
ulty of Pharmacy, Airlangga University Jl. Dharmawangsa dalam, Surabaya
60286, Indonesia.
Betamethasone dipropionate (Welding, Hamburg, Germany;
Batch BDP005/0502; Assay 99.4%) and salicylic acid (PT
BrataChem, Surabaya, Indonesia; Batch 229/SC/MP/92; Assay
99.5%) were pharmaceutical-grade substances. The substances
fulfilled the requirement of the Indonesian Pharmacopoeia,
1995 [3], and were used as received for preparing laboratory-
made lotion and standard solutions.
Chloroform, 96% ethanol, dichloromethane (E. Merck), toluene
(Riedel–de Haen), and glacial acetic acid (J.T. Baker) were ana-
lytical-grade reagents; solvents and reagents were used without
further purification.
Excipients for preparation of laboratory-made lotion were phar-
maceutical-grade substances; lotion was prepared by mixing
propylene glycol (20 g), nipagin (150 mg), and glycerol (30 g)
and adjusting the volume to 100 mL with 96% ethanol. Lotions
were prepared containing five different concentrations of
betamethasone dipropionate (52.0, 58.5, 65.0, 71.5, and
78.0 mg/100 mL) and salicylic acid (1600, 1800, 2000, 2200 and
2400 mg/100 mL). A commercial lotion, produced in Indonesia

438 VOL. 16. NOVEMBER/DECEMBER 2003

DOI: 10.1556/JPC.16.2003.6.6
Journal of Planar Chromatography
 Analysis of Betamethasone Dipropionate and Salicylic Acid in Lotions

and containing betamethasone dipropionate (65 mg/100 mL)

and salicylic acid (2000 mg/100 mL) was purchased from a
local Pharmacy in Surabaya in February 2003.
Stock standard solutions were prepared daily by dissolving
accurately weighed betamethasone dipropionate (325.0 mg) in
96% ethanol (10.0 mL); 2.0 mL of this solution was added to a
solution of 400.0 mg salicylic acid (accurate weight) in ca
45 mL 96% ethanol in a 50.0-mL volumetric flask and the
resulting solution was diluted to volume with 96% ethanol. Dif-
ferent standard solutions were prepared from the stock solution
by dilution with 96% ethanol. For linearity studies, solutions
were prepared containing 400, 800, 1300, 1600, 2000, 2400,
2700, 3200, 3555, and 4000 µg mL–1 salicylic acid and 65, 130,
220, 260, 325, 390, 430, 520, 580 and 650 µg mL–1 betametha-
sone dipropionate. These solutions (1.0 µL) were spotted on the Figure 1
TLC plate. In-situ absorbance/reflectance spectrum of betamethasone dipropionate spot
from λ = 200 to 400 nm, with its λmax (at 250 nm). Stationary phase: precoated sil-
ica gel 60 F254 plate (E. Merck); mobile phase: ethanol (96%)–toluene–chloro-
2.2 Sample Preparation form–glacial acetic acid, 6.0 + 20 + 14 + 0.5 (v/v).

A sample of lotion (1.00 mL) was transferred to a 10.0-mL vol-

umetric flask and diluted to volume with 96% ethanol. The solu-
tion obtained (1.0 µL for analysis of salicylic acid and
5.0 µL for analysis of betamethasone dipropionate) was spotted
on a TLC plate together with standards.

2.3 Chromatography

Chromatography was performed on precoated silica gel 60

F254 plates (E. Merck #1.05554) which were cut into 10 cm ×
20 cm plates before use); a Nanomat III (Camag, Muttenz,
Switzerland) was used for sample application. The distance
from the lower edge was 10 mm, the distance from the side
was 15 mm, and the distance between tracks was 10 mm. The
mobile phase used in this experiment was ethanol (96%)–
toluene–chloroform–glacial acetic acid, 6.0 + 20 + 14 + 0.5
Figure 2
(v/v). Ascending development was performed in a Camag
In situ absorbance/reflectance spectrum of salicylic acid spot from λ = 200 to
twin-trough chamber (for 20 cm × 10 cm plates) after satura- 400 nm, with its λmax (at 310 nm). Stationary phase: precoated silica gel 60 F254
tion for at least 2 h; in all experiments the mobile-phase migra- plate (E. Merck); mobile phase: ethanol (96%)–toluene–chloroform–glacial acetic
tion distance was 8.0 cm. (development time ca. 30 min at acid, 6.0 + 20 + 14 + 0.5 (v/v).

25 ± 2°C).
Densitometric scanning was performed with a Camag TLC-
Scanner II. The purity and identity of the analyte spots were
determined by scanning in absorbance/reflectance mode from
λ = 200 to 400 nm. Quantitative evaluation was performed by
measuring the absorbance/reflectance of the analyte spots at λ =
250 nm for betamethasone dipropionate and λ = 310 nm for sal-
icylic acid (Figures 1 and 2). The densitometric scanning set-
tings were: bandwidth 10 nm, slit width 4, slit length 6, and
scanning speed 4 mm s–1. Calculations for identity, purity
checks (rS,M and rM,E where S = start, M = center, E = end spec-
trum), sdv (relative standard deviation) of the linear/calibration
plot, and quantification of the analyte spots were performed by
use of CATS version 3.17 (1995) software (Camag). Routine
quantitative evaluation was performed on the basis of peak area
with linear regression, using 4 or 5 calibration points for each
plate (80 to 120% of the expected value).
2.4 Validation
The method was validated for linearity, homogeneity, detection
limit (DL), accuracy, and range by a modification of the method
of Funk et al. [11] and Hahn-Dienstrop [12]. The selectivity of
the method was proved by identification and purity checks of
the analyte spots. A five-point accuracy study (80, 90, 100, 110,
and 120% of the expected value) was performed for the labora-
tory-made lotions. For commercial preparations accuracy stud-
ies were performed by use of single-point standard addition
(40% of label claim). The precision (repeatability and interme-
diate precision) was evaluated by analyzing six different extract
aliquots from laboratory-made lotions containing 52.0, 65.0, or
78.0 mg betamethasone dipropionate/100 mL and 1600, 2000,
or 2400 mg salicylic acid/100 mL by a modification of the
method of Renger et al. [13].

Journal of Planar Chromatography VOL. 16. NOVEMBER/DECEMBER 2003 439

Analysis of Betamethasone Dipropionate and Salicylic Acid in Lotions
3 Results and Discussion
After development of the TLC plate the densitogram obtained at
λ = 250 nm (Figure 3) contained the peaks of salicylic acid
(RF 0.30), nipagin (RF 0.40), and betamethasone dipropionate
(RF 0.52) whereas the densitogram obtained at λ = 310 nm con-
tained the spot of salicylic acid only. Although by scanning at
λ = 250 nm all the analyte spots could be detected, and shown to
be well separated, severe fronting was observed for the salicylic
acid spot and its tailing factor (T) was approximately 0.36 at
10% of peak height. If scanning was performed at λ = 310 nm
the peak shape was better (T = 0.68). Our preliminary work
showed that scanning at λ = 250 nm resulted in poor results val-
ues of the sdv of the calibration plot and RSD from study of the
precision of salicylic acid measurement (>5 and >2%, respec-
tively), so for further work the TLC plates were scanned twice
consecutively using two different wavelengths–250 nm for
betamethasone dipropionate and 310 nm for salicylic acid. Use
of this TLC system revealed that all analyte spots of the labora-
tory-made lotions and commercial preparations furnished in-
situ UV spectra identical with those of standards (r ≥ 0.9999).
A check of the purity of the analyte spots using CATS software
also showed that all analyte spots obtained from the extracts
were pure. The values of rS,M and rM,E were ≥0.9999, showing
the proposed TLC method is highly selective. Our recent work
has also shown that if the chloroform was replaced by
dichloromethane the profile of the densitogram was almost
identical (data not shown). In this work chloroform was used
because of the limited supply of dichloromethane in our labora-
tory at the time validation was performed.
Peak area was observed to be linearly dependent on the amount
of betamethasone dipropionate within the range 65 to 650 ng
spot–1. The relative process standard deviation, VXO [11] was
3.2% (the linear regression equation for the calibration plot was
Y = 34.9 + 2.50X; n = 10; sdv = 2.7; r = 0.9985). The calculated
value of the test parameter Xp (for p = 0.05) [11] was satisfacto-
ry (50 ng spot–1). For salicylic acid spots in the range 400 to
Figure 3
Densitograms (λ = 250 nm) obtained from: (1) solution of standards of
betamethasone dipropionate and salicylic acid; (2) extract of laboratory-made
lotion; (3) extract of excipients of laboratory-made lotion; (4) extract of commer-
cial lotion. Peak identities: (A) salicylic acid; (B) nipagin; (C) betamethasone
dipropionate.
440 VOL. 16. NOVEMBER/DECEMBER 2003
Figure 4
Densitograms (λ = 310 nm) obtained from: (1) solution of standard salicylic acid;
(2) extract of laboratory-made lotion; (3) extract of excipients of laboratory-made
lotion; (4) extract of commercial lotion.
4000 ng spot–1 a polynomial regression line was obtained (the
equation for the calibration plot was Y = 576.1 + 1.069X –
0.0001X2; n = 10, sdv = 2.0, Vxo = 5.1%). When the concentra-
tion range of salicylic acid was reduced (1600 to 2400 ng spot–1)
a linear relationship was obtained (linear regression equation
Y = 1255 + 0.757X; n = 7, sdv = 1.5, Vxo = 2.0%, r = 0.9901). The
calculated value of test parameter Xp (for p = 0.05) [11] was sat-
isfactory (472 ng spot–1). The ANOVA regression test for linear-
ity of the two regression lines resulted in significant calculated
F values (betamethasone dipropionate 2692.8, salicylic acid
250.5; p < 0.0001). Plots of the residuals against quantity of
analyte confirmed the linearity of the basic calibration graphs
(data not shown). The residuals were distributed at random
around the regression lines; neither trend nor uni-directional
tendency was found.
The basic linear calibration plots showed variance homogeneity
over the whole range. The calculated test values PW [11] were
1.32 (salicylic acid) and 6.00 (betamethasone dipropionate).
The PW values were less than the Ftable value (6.03 for f1 = 8,
f2 = 8; p = 0.01).
Although the validation data DL and QL (quantitation limit)
were not required for assay of the active ingredient(s) in lotions,
they were also determined, because they might be useful for
other purposes (e.g. in-vitro bio-equivalence and stability stud-
ies, etc.). DL was determined by performing linear regression
for relatively low concentrations of betamethasone dipropionate
(16 to 65 ng spot–1) according to the method of Funk et al. [11].
The calculated equation of the regression line was Y = 7.13 +
10.96X (n = 6; VXO = 4.7%; r = 0.9958 ; Fcalculated value = 467.5
for p < 0.0001). The calculated value of Xp (for p = 0.05) [11]
was 10.7 ng spot–1. In this case DL = Xp [11]. For salicylic acid
the concentration ranges were 32 to 200 ng spot–1; in this range
the equation was Y = 86.42 + 4.62X (n = 5; r = 0.9977; Vxo =
5.8%; Fcalculated value = 640.6 for p < 0.0001) and the value of Xp
was 30.6 ng spot–1. According to Carr and Wahlich [14] the
value of the QL could be estimated as three times the DL value.
Journal of Planar Chromatography
 Analysis of Betamethasone Dipropionate and Salicylic Acid in Lotions

Table 1 {VB(bf)} from the recovery plots revealed no occurrence of

Results from determination of the accuracy of analysis of laboratory- constant and proportional systematic errors [11].
made lotion. This work also showed that all the SD values from evaluations of
No. Betamethasone Salicylic acid precision (data not shown) were <1/6 [15] of the specification
dipropionate [ng spot–1] [ng spot–1] range of betamethasone dipropionate and salicylic acid in topical
Xc a) Xf b) Xc a) Xf b) preparations [3]. All RSD obtained from evaluations of repeatabil-
ity and intermediate precision had values <2% (Table 2). Mea-
1 260 260 1600 1641 surements 1–3 were performed in one laboratory by different ana-
2 293 284 1800 1803 lysts, on different plates and days, on three different concentra-
3 325 318 2000 2025 tions of the analytes in the laboratory-made lotions. These results
demonstrated that the accuracy and precision of the proposed
4 357 349 2200 2204
method were satisfactory in the range of 80 to 120% of the expect-
5 390 382 2400 2452 ed concentration. The proposed TLC method is, therefore, suitable
Mean recovery ± SD [%] 98.1 ± 1.1 101.3 ± 1.1
for the routine analysis of products of similar composition in phar-
maceutical industry quality-control laboratories.
Recovery curve Xf = 8.71 + 0.95Xc Xf = 1.99 + 1.01Xc
VB(af) c) 8.71 ± 32.51 1.99 ± 252.8
VB(bf ) c) 0.95 ± 0.09 1.01 ± 0.12
a)Nominal concentration of the analyte in the spotted solution References
b)Measured concentration of the analyte in the spotted solution [1] Informasi Spesialite Obat Indonesia (ISO), Ikatan Sarjana Farmasi
c)For α = 0.05 Indonesia, Jakarta, Indonesia, Volume XXXV, 2001, p. 390.
[2] The United States Pharmacopoeia 25—NF 20Asian edn, United
Table 2 States Pharmacopeial Convention, Rockville, MD, 2002, pp.
Results from determination of the precision of analysis of laboratory-
224–226, 1548–1550.
made lotion. [3] Farmakope Indonesia, Edisi IV (Indonesian Pharmacopoeia, 4th
edn), Departemen Kesehatan Republik Indonesia, Jakarta, 1995,
Measurement RSD [%; n = 6]a) pp 51–52, 138–140.
Betamethasone Salicylic acid
dipropionate [mg/100 mL] [4] British Pharmacopoeia 2000, Volume I, The Stationary Office,
[mg/100 mL] London, 2000, pp. 203–204.
52.0 65.0 78.0 1600 2000 2400 [5] Pharmacopoeia of the People’s Republic of China, Vol. II, English
edn, 1997, Chemical Industry Press, Beijing, 1997, pp. 535–536.
1 1.81 1.42 1.46 1.21 1.32 0.80
[6] Indian Pharmacopoeia 1996, Vol. II, The Controller of Publica-
2 1.21 0.80 1.13 1.78 0.54 1.05 tions, Delhi, 1996, p. 673.
3 1.86 1.27 ndb) 0.79 nd nd [7] British Pharmacopoeia 2000, Volume II, The Stationary Office
a)Each London, 2000, pp. 2220–2221.
measurement was performed by a different analyst on different
days [8] E. R. Kedor-Hackmann, E.A. Gianotto, and M. I. Santoro, Drug.
b)Not determined Dev. Ind. Pharm. 24 (1998) 553–555.
[9 ] Camag Bibliography Service, Cumulative CD version 1.04,
For determination of analytes in laboratory-made lotions or Camag, Muttenz, 2001.
commercial preparations the amounts of solutions spotted were [10] K. Datta and S.K. Das, J. Assoc. Off. Anal. Chem. 77 (1994)
different for betamethasone dipropionate (5.0 µL) and salicylic 1435–1438.
acid (1.0 µL), because of the large difference between the sali- [11] W. Funk, V. Damman, and G. Donnervert, Qualitätssicherung in
cylic acid (2000 mg/100 mL) and betamethasone dipropionate der Analytischen Chemie, VCH, Weinheim, 1992, pp. 12–36,
(65 mg/100 mL) content. Table 1 shows that accuracy, as 161–180.
revealed by the mean recovery data [%] for the laboratory-made [12] E. Hahn-Dienstrop, Applied Thin Layer Chromatography,
lotion (98.1 and 101.3%), was good. Mean recovery data Wiley–VCH, Weinheim, 2000, pp. 198–205.
obtained by use of the standard addition method on the com-
[13] B. Renger, H. Jehle, M. Fischer, and W, Funk, J. Planar Chro-
mercial preparation were also good (99.8% for betamethasone matogr. 8 (1995) 269–278 (1995).
dipropionate; 98.5% for salicylic acid; n = 2). To prove system-
atic errors did not occur, linear regression was performed on the [14] G.P. Carr and J.C. Wahlich, J. Pharm. Biomed. Anal. 8 (1990)
613–618.
recovery plot of Xf (concentration of the analyte measured by
the proposed method) against Xc (nominal concentration of the [15] J. Ermer, J. Pharm. Biomed. Anal. 24 (2001) 755–767.
analyte) constructed for the laboratory-made lotions. The confi- Ms received: June 12, 2003
dence range data (p = 0.05) of the intercept {VB(af)} and slope Accepted by SN: September 9, 2003

Journal of Planar Chromatography VOL. 16. NOVEMBER/DECEMBER 2003 441

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