American Journal of Analytical Chemistry, 2010, 1, 113-126
doi:10.4236/ajac.2010.13015 Published Online November 2010 (http://www.SciRP.org/journal/ajac)
A Validated Stability-Indicating LC Method for
Fluocinonide in the Presence of Degradation Products,
Its Process-Related Impurities and Identification of
Degradation Products
Prabha Srinivasu1,2, Devarakonda V. SubbaRao1, Raju V. K. Vegesna1, K. Sudhakar Babu2
1Versapharm Incorporated, Warminister, USA
2Department of Chemistry, Sri Krishnadevaraya University, Anantapur, India
Received July 26, 2010; revised October 14, 2010; accepted October 27, 2010
Abstract
The objective of the current study was to develop a validated, specific and stability-indicating reverse phase
liquid chromatographic method for the quantitative determination of fluocinonide and its related substances.
The determination was done for active pharmaceutical ingredient and its pharmaceutical dosage forms in the
presence of degradation products, and its process-related impurities. The drug was subjected to stress condi-
tions of hydrolysis (acid and base), oxidation, photolysis and thermal degradation per International Confer-
ence on Harmonization (ICH) prescribed stress conditions to show the stability-indicating power of the
method. Significant degradation was observed during acid, base hydrolysis, and peroxide degradation. The
major degradants were identified by LC-MS, FTIR and 1H/13C NMR spectral analysis. The chromatographic
conditions were optimized using an impurity-spiked solution and the samples generated from forced degra-
dation studies. In the developed HPLC method, the resolution between fluocinonide and its process-related
impurities, (namely imp-1, imp-2, imp-3, imp-4, imp-5, imp-6, imp-7 and imp-8) and its degradation products
was found to be greater than 2.0. The chromatographic separation was achieved on a C18, 250 mm × 4.6 mm,
5 µm column. The LC method employed a linear gradient elution and the detection wavelength was set at
240 nm. The stress samples were assayed against a qualified reference standard and the mass balance was
found to be close to 99.3%. The developed RP-LC method was validated with respect to linearity, accuracy,
precision and robustness.
Keywords: Fluocinonide, RP-LC, LC-MS, Forced Degradation, Validation, Stability-Indicating
1. Introduction
Fluocinonide, pregna-1, 4-diene-3, 20-dione, 21-(acety-
loxy)-6, 9-difluoro-11-hydroxy-16, 17-[(1-methylethyli-
dene)bis(oxy)]-, (6α, 11β, 16α)-, is a potent glucorticoid
steroid used topically as anti-inflammatory agent for the
treatment of skin disorders such as eczema. It relieves
itching, dryness, redness, crusting, scaling, inflammation
and discomfort. Fluocinonide is a white or almost white
crystalline powder. It is practically insoluble in water;
slightly soluble in absolute ethanol. There are seven
classes of topical corticosteroids ranked according to
their potency based vasoconstrictor assays. Fluocinonide
is a fluorinated, highly potent, corticosteroid. It ranks as
a “high-potency” (second-highest rank) topical cortico-
steroid [1]. Corticosteroids have multiple actions, in-
cludeing anti-inflammatory and anti-proliferative effects.
When applied topically for at least one week, fluocinon-
ide is effective in the treatment of inflammation and
itching caused by a number of skin conditions such as
allergic reactions, eczema and psoriasis. Minimal amounts
should be used for a minimal length of time to avoid the
occurrence of adverse effects. Fluocinonide is also used
in veterinary medicine. It is used for treatment of aller-
gies in dogs. Natural systemic cortisol concentrations can
be suppressed for weeks after one week of topical expo-
sure.
A chromatographic assay method has appeared in US

114 P. SRINIVASU ET AL.
pharmacopeia for the quantification of fluocinonide in
drug substance and its pharmaceutical topical dosage
forms [2]. Few analytical methods were available in lit-
erature for the quantification of fluocinonide and fluclo-
cortisone acetate. Shek E. et al. has described an HPLC
procedure to chromatograph fluocinonide as well as tri-
amcinolone acetonide and diflorasone diacetate [3-6]. In
addition, Shek E. et al. reported the stability indicating
LC method for triple corticoid integrated system in a
cream. [3] in which Shek. E. et al. described the quanti-
fication of–fluocinonide, procinonide and ciprocinonide
and expected the major degradation product under hy-
drolytic conditions would be fluocinolone acetonide.
Further study was not carried out to confirm this impu-
rity. The present study was carried out to describe de-
tailed degradation studies as per ICH guidelines and de-
veloped a suitable LC method for the separation and es-
timation of both process related impurities and degrada-
tion impurities. Interestingly author observed two major
degradation products in acid, base hydrolysis and perox-
ide degradation of fluocinonide. The major degradation
products of fluocinonide were isolated using preparative
HPLC and structure elucidation was performed for the
same using advanced spectral techniques such as NMR,
LC-MS and IR. The purpose of the present research
work was to develop a suitable single stability-indicating
LC method for the determination of fluocinonide and its
related substances and structure elucidation of major
degradation products. The developed LC method was
validated with respect to specificity, LOD, LOQ, linear-
ity, precision, accuracy and robustness. Forced degrada-
tion studies were performed on the drug substance and
drug products to show the stability-indicating nature of
the method [7-13]. These studies were performed in ac-
cordance with established ICH guidelines [14-15].
2. Experimental
2.1. Chemicals
Samples of fluocinonide and its related impurities were
received from Versapharm Incorporated, Warminister,
PA, USA. (Figure 1). All of the impurities and the fluo-
cinonide standard were of > 90% purity and are as fol-
lows:-, fluocinonide (99.6%), imp-1 (95.9%), imp-2
(99.3%), imp-3 (93.7%), imp-4 (99.5%) imp-5 (96.7%),
imp-6 (98.5%), imp-7 (97.8%) and imp-8 (99.2%).
Commercially available fluocinonide ointment and fluo-
cinonide solution (LIDEX) were purchased for this study.
In addition, HPLC grade acetonitrile and methanol were
purchased from Merck, (Darmstadt, Germany). Analyti-
cal reagent grade sodium dihydrogen phosphate mono-
hydrate, phosphoric acid and acetic acid were purchased
Fluocinonide
1
2
3
4
5
10
O
67
8
914
13
12
11
15
16
17
F
F
HO
19
18
20
O
21
O
O
O
24
26
25
O
22 23
(a) pregna-1, 4-diene-3, 20-dione, 21-(acetyloxy)-6, 9-difluoro-11-
hydroxy-16, 17-[(1-methylethylidene)bis(oxy)]-, (6α, 11β, 16α)-. Mo-
lecular weight: 494.52.
Imp-1
1
2
3
4
5
10
O
67
8
9
14
13
12
11
15
16
17
F
F
HO
19
18
20
O
21
OH
OH
OH
(b) pregna-1, 4-diene-3, 20-dione, -6, 9-difluoro-11, 16, 17, 21-
tetrahydroxy-, (6α, 11β, 16α)-. Molecular weight: 412.42.
Imp-2
1
2
3
4
5
10
O67
8
914
13
12
11
15
16
17
F
F
HO
19
18
20 O
21
OH
O
O
23
25
24
H3CO
22
(c) pregna-1, 4-diene-3, 20-dione, -6, 9-difluoro-11, 21-dihydroxy-
21methoxy-16, 17-[(1-methylethylidene)bis(oxy)]-(6α, 11β, 16α) Mo-
lecular weight: 482.51.

P. SRINIVASU ET AL.
115
Imp-3
1
2
3
4
5
10
O67
8
914
13
12
11
15
16
17
F
F
HO
19
18
20 O
21
OH
O
O
22
24
23
(d) pregna-1, 4-diene-3, 20-dione, -6, 9-difluoro-11, 21-dihydroxy-16,
17-[(1-methylethylidene)bis(oxy)]-, (6β, 11β, 16α)-. Molecular weight:
452.49.
Imp-4
1
2
3
4
5
10
O
67
8
914
13
12
11
15
16
17
F
19
18
20
O
21
OH
O
O
22
24
23
O
(e) pregna-1, 4-diene-3, 20-dione, -6-fluoro-9, 11 epoxy, -21-
hydroxy-16, 17-[(1-methylethylidene)bis(oxy)]-, (6α, 11β, 16α)-. Mo-
lecular weight: 432.48.
Imp-5
1
2
3
4
5
10
O
67
8
914
13
12
11
15
16
17
Cl
F
HO
19
18
20
O
21
OH
O
O
22
24
23
(f) pregna-1, 4-diene-3, 20-dione, -6 chloro, 9-fluoro-11, 21-dihydroxy-
16, 17-[(1-methylethylidene)bis(oxy)]-, (6α, 11β, 16α)-. Molecular
weight: 468.94.
Imp-6
1
2
3
4
5
10
O
67
8
914
13
12
11
15
16
17
F
F
HO
19
18
20
O
21
O
O
O
24
26
25
O
22 23
(g) pregna-1, 4-diene-3, 20-dione, 21-(acetyloxy)-6, 9-difluoro-11-
hydroxy-16, 17-[(1-methylethylidene)bis(oxy)]-, (6β, 11β, 16α)-. Mo-
lecular weight: 494.52.
Imp-7
1
2
345
10
O
6
7
8
914
13
12
11
15
16
17
F
HO
19
18
20
O
21
O
O
O
24
26
25
O
22 23
F
(h) pregna-1, 4-diene-3, 20-dione, 21-(acetyloxy)-4, 9-difluoro-11-
hydroxy-16, 17-[(1-methylethylidene)bis(oxy)]-, (4α, 11β, 16α)- Mo-
lecular weight: 496.54.
Imp-8
1
2
3
4
5
10
O
67
8
914
13
12
11
15
16
17
C
l
F
HO
19
18
20
O
21
O
O
O
24
26
25
O
(i) pregna-1, 4-diene-3, 20-dione, 21-(acetyloxy)-6-Chloro, 9-fluoro-
11-hydroxy-16, 17-[(1-methylethylidene)bis(oxy)]-, (6α, 11β, 16α)-. Mo-
lecular weight: 510.18.

116 P. SRINIVASU ET AL.
Degradation (Base degradation) impurity at RRT 0.54
1
2
3
4
5
10
O
67
8
9
14
13
12
11
15
16
17
F
F
HO
19
18
20
O
21
O
OH
OH
22
O
23
(j) Pregna-1, 4-diene-3, 20-dione, 21-(acetyloxy)-6, 9-difluoro-11, 16,
17-trihydroxy-(6α, 11β, 16α)- Molecular weight: 454.46.
Degra dat i on (A cid an d per oxi de degr ad at ion ) im purit y at RRT 0. 6 0
1
2
3
4
5
10
O
67
8
914
13
12
11
15
16
17
F
F
HO
19
18
20
O
21
OH
O
O
22
23
24
(k) Pregna-1, 4-diene-3, 20-dione, 6, 9-difluoro-11, 21 dihydroxy-16,
17-[(1-Methylethylidene) bis (oxy)]-, (6α, 11β, 16α)- Molecular weight:
452.49.
Figure 1. Structures and names of fluocinonide and its im-
purities.
from Merck. Highly pure water was prepared with the
Millipore Milli-Q Plus water purification system.
2.2. Equipment
The LC system used for method development, forced
degradation studies and method validation consisted of a
Waters 2695 binary pump with an auto sampler and a
2996 photo diode array detector (PDA). The output sig-
nal was monitored and processed using Empower soft-
ware on a Pentium computer (Digital equipment Co.).
Photo stability studies were carried out in a photo stabi-
lity chamber (Sanyo, Leicestershire, UK). Thermal stabi-
lity studies were carried out in a dry air oven (Lindberg-
Blue, USA).
2.3. Chromatographic Conditions
A Waters symmetry C18 250 mm × 4.6 mm, 5 µm col-
umn was used with a mobile phase containing a gradient
of solvents A and B. Solvent A was composed of water,
with its pH adjusted to 3.0 with orthophosphoric acid.
Water and acetonitrile in the ratio of 150:850 (v/v) was
used as solvent B. The flow rate of the mobile phase was
1.0 ml/min with a gradient program of 0/40, 30/80, 35/80,
35.1/40 and 45/40 (time (min)/%B). The column tem-
perature was maintained at 27°C and the detection
wavelength was set at 240 nm. The injection volume was
20 μl. The diluent consisted of water and acetonitrile in a
ratio of 50:50 (v/v).
2.4. LC-MS Conditions
The LC-MS system (Agilent 1100 series liquid chroma-
tography system coupled with a 6400 series triple quad-
rapole mass spectrometer) was used for the identification
of unknown compounds formed during forced degrada
tion. A Waters symmetry C18 250 mm × 4.6 mm, 5 µm
column was used as the stationary phase. Water, acetone-
trile and acetic acid in a ratio of 60:40:1 (v/v/v) was used
as the mobile phase. A mixture of water and acetonitrile
in a 50:50 (v/v) ratio was used as the diluent. The flow
rate was 1.0 ml/min. The analysis was performed in
positive and negative electrospray ionization modes. The
capillary and cone voltages were 3.5 kV and 25 V, re-
spectively. The source and dissolvation temperatures
were 120°C and 350°C, respectively and the dissolvation
gas flow was 500 l h-1.
2.5. Preparation of Standard Solutions and
Sample Solutions
A stock solution of fluocinonide (2.5 mg/ml) was pre-
pared by dissolving the appropriate amount of fluoci-
nonide solid in the diluent. Working solutions of 250 and
25 μg/ml were prepared from the stock solution for the
determinations of related substances and assay respect-
tively. A stock solution of impurity (mixture of imp-1,
imp-2, imp-3, imp-4, imp-5, imp-6, imp-7 and imp-8) at
0.25 mg/ml was also prepared in the diluent.
Fluocinonide solution sample preparation: The solution
(2.5 g) equivalent to 2.5 mg of drug was transferred into
a 10 ml volumetric flask, and 2 ml of diluent was added.
The flask was attached to a rotary shaker and shaken for
10 min to mix completely. The mixture was sonicated for
2 min and then diluted to the appropriate volume with
diluent to make a solution containing 0.25 mg/ml. The
solution was filtered through a 0.45 µ Nylon 66 mem-
brane filter.

P. SRINIVASU ET AL.
117
Fluocinonide ointment sample preparation: Sample
(2.5 g ointment) equivalent to 2.5 mg of drug was trans-
ferred into a 50 ml screw test tube, and 5 ml of diluent
was added. The test tube was heated for 5 min at 65°C,
cooled to room temperature, cyclomix for 2 min and re-
peated the procedure once again, cool to room temp and
added 5ml of diluent cyclomix for 3 min. The solution
was filtered through a 0.45 µ Nylon 66 membrane filter.
These solutions were used for the related substances es-
timation of fluocinonide. For assay analysis 19-nore-
thindrone was used as internal standard.
2.6. Stress Studies/Specificity
Specificity is the ability of the method to measure the
analyte response in the presence of its potential impuri-
ties [14]. The specificity of the developed LC method for
fluocinonide was determined in the presence of its impu-
rities (namely imp-1, imp-2, imp-3, imp-4, imp-5, imp-6,
imp-7 and imp-8) and degradation products. Forced deg-
radation studies were also performed on fluocinonide to
provide an indication of the stability- indicating property
and specificity of the proposed method [7-12]. The stress
conditions employed for the degradation study included
light (carried out as per ICH Q1B), heat (60°C), acid
hydrolysis (1M HCl), base hydrolysis (0.1M NaOH) and
oxidation (5% H2O2). For heat and light studies, the
samples were exposed for 10 days, whereas the samples
were treated for 48 h for acid hydrolysis and for oxida-
tion, 30 minutes for base hydrolysis. The peak purity of
the fluocinonide stressed samples was checked by using
a Waters 2996 photo diode array detector (PDA). The
purity angle was within the purity threshold limit in all of
the stressed samples, demonstrating the homogeneity of
the analyte peak.
Assays were carried out for the stress samples against
a qualified reference standard. The mass balance (% as-
say + % of impurities + % of degradation products) was
calculated for all of the samples.
2.7. Method Validation
The proposed method was validated per ICH guide lines
[14-15].
2.7.1. Precision
The precision of the related substance method was investi-
gated by injecting six individual preparations of (250 μg/ml)
fluocinonide spiked with 0.15% each of imp-1, imp-2,
imp-3, imp-4, imp-5, imp-6, imp-7 and imp-8. The %RSD
of the areas of each imp-1, imp-2, imp-3, imp-4, imp-5,
imp-6, imp-7 and imp-8 was calculated.
The intermediate precision of the method was evalu-
ated using a different analyst and instrument located
within the same laboratory.
The precision of the assay method was evaluated by
carrying out six independent assays of a test sample of
fluocinonide against a qualified reference standard. The
%RSD of six obtained assay values was calculated.
2.7.2. Limit of Detection (LOD) and Limit of
Quantification (LOQ)
The LOD and LOQ for imp-1, imp-2, imp-3, imp-4,
imp-5, imp-6, imp-7 and imp-8 were estimated at a sig-
nal-to-noise ratio of 3:1 and 10:1, respectively, by in-
jecting a series of dilute solutions with known concentra-
tions. The precision study was also carried at the LOQ
level by injecting six individual preparations of imp-1,
imp-2, imp-3, imp-4, imp-5, imp-6, imp-7 and imp-8 and
calculating the %RSD of the areas.
2.7.3. Linearity
Linearity test solutions for the assay method were pre-
pared from a stock solution at five concentration levels
from 50 to 150% of the assay analyte concentration (12.5,
20, 25, 30, and 37.5 μg/ml). The peak area versus con-
centration data was analyzed with least-squares linear
regression.
Linearity test solutions for the related substance
method were prepared by diluting the impurity stock
solution (2.5) to the required concentrations. The solu-
tions were prepared at eight concentration levels from
the LOQ to 150% of the specification level (LOQ, 0.05%,
0.10%, 0.15%, 0.20%, 0.25% and 0.3%). The slope and
y-intercept of the calibration curve are reported.
2.7.4. Accuracy
The accuracy of the assay method was evaluated in trip-
licate at four concentration levels (LOQ, 12.5, 25 and
37.5 μg/ml), and the percentage recoveries were calcu-
lated.
The drug substance did not show the presence of
imp-1, imp-2, imp-3, imp-4, imp-5, imp-7 and imp-8, but
contained 0.29% of imp-6. Standard addition and recov-
ery experiments were conducted to determine the accu-
racy of the related substance method for the quantifica-
tion of all eight impurities (imp-1, imp-2, imp-3, imp-4,
imp-5, imp-6, imp-7 and imp-8) in the drug substance as
well as in the drug product. The study was carried out in
triplicate at 0.075%, 0.15% and 0.225% of the analyte
concentration (250 μg/ml). The percentage of recoveries
for imp-1, imp-2, imp-3, imp-4, imp-5, imp-6, imp-7 and
imp-8 were calculated.
2.7.5. Robus tness
To determine the robustness of the developed method,

P. SRINIVASU ET AL.
118
the experimental conditions were altered and the resolu-
tion between fluocinonide and imp-1, imp-2, imp-3,
imp-4, imp-5, imp-6, imp-7 and imp-8 was evaluated.
The flow rate of the mobile phase was 1.0 ml/min. To
study the effect of the flow rate on the resolution, the
flow rate was changed by 0.1 units (0.9 and 1.1 ml/min).
The effect of pH on the resolution of the impurities was
studied by varying the pH by ± 0.1 units (buffer pH of
2.9 and 3.1). The effect of the column temperature on the
resolution was studied at 22°C and 32°C instead of 27°C.
In all these varied conditions, the components of the mo-
bile phase remained constant, as outlined in Subsection 2.3.
2.7.6. Solution Stability and Mobile Phase Stability
The solution stability of fluocinonide in the assay
method was carried out by leaving both the sample and
reference standard solutions in tightly capped volumetric
flasks at room temperature for 48h. The same sample
solutions were assayed for in 6h intervals over the study
period. The mobile phase stability was also examined by
assaying the freshly prepared sample solutions against
freshly prepared reference standard solutions for 6h in-
tervals up to 48h. The prepared mobile phase remained
constant during the study period. The %RSD of the fluo-
cinonide assay was calculated for the mobile phase and
solution stability experiments.
The solution stability of fluocinonide and its impuri-
ties in the related substance method was carried out by
leaving a spiked sample solution in a tightly capped
volumetric flask at room temperature for 48h. The con-
tent of imp-1, imp-2, imp-3, imp-4, imp-5, imp-6, imp-7
and imp-8 was determined at 6h intervals up to the study
period.
The mobile phase stability was also investigated for
48h by injecting the freshly prepared sample solutions
for every 6h interval. The content of imp-1, imp-2, imp-3,
imp-4, imp-5, imp-6, imp-7 and imp-8 was determined in
the test solutions. The prepared mobile phase remained
constant during the study period.
3. Results and Discussion
3.1. Method Development and Optimization
All the impurities and fluocinonide solutions were pre-
pared in diluent at a concentration of 10 ppm and
scanned in UV-visible spectrophotometer; all the eight
impurities and fluocinonide had a UV maxima at around
240 nm (Figure 2). Hence detection at 240 nm was se-
lected for method development purpose.
The main objective of the chromatographic method
was to separate imp-1, imp-2, imp-3, imp-4, imp-5,
imp-6, imp-7, imp-8 and the generated degradation
products from the analyte peak during stress studies.
Impurities and degradation products were co-eluted by
using different stationary phases, such as C8, cyano and
phenyl with various mobile phases with buffers, such as
phosphate and acetate with different pH values (3-7), and
organic modifiers, including acetonitrile and methanol.
United States Pharmacopeia stated LC method for the
Fluocinonide imp-1 imp-2 imp-3 imp-4 imp-5 imp-6 imp-7 imp-8
Figure 2. Typical spectrums of fluocinonide and its impurities.

P. SRINIVASU ET AL.
119
estimation of related substances of fluocinonide for drug
substance [2]. Hence this was chosen as initial experi-
ment. The method consists of water and acetonitrile
(50:50, v/v) as mobile phase at a flow rate of 2.0 ml/min
with Waters bonda pack 300 mm × 3.9 mm ID column
and 10 µm particle size C18 stationary phase. When an
impurity spiked solution of fluocinonide was injected.
Imp-1 and Imp-2 were eluted at void with retention times
of 0.8 and 1.3, imp-6 and imp-7 were co eluted (Figure
3). In acid degradation sample the resolution between
one of the major degradant and imp-3 was very less (Rs <
1.2). To improve the resolution between impurities and
retention of imp-1 and imp-2, particle size of the column
was decreased to 5 µm and flow rate was decreased to
1.0 ml/min and injected the impurity spiked solution.
The resolution between Imp-6 and imp-7 was slightly
improved but there is no improvement in the retention of
imp-1 and imp-2. To improve the retention times of
imp-1 and imp-2 acetonitrile content in mobile phase
was decreased to 30% (water: acetonitrile: 70:30) and
injected impurity spiked solution. The retention of imp-1
and imp-2 was improved but fluocinonide peak was
eluted at round 60 min. Isocratic trails were not successful
in achieving a favorable resolution between the impuri-
ties and retention of imp-1, imp-2 and fluocinonide.
Therefore, a gradient method was selected using buffer
(0.01M ammonium acetate, pH 7.0) as mobile phase A
and water and acetonitrile in a ratio of 100:900, v/v as
mobile phase B, retention time of imp-1 and imp-2 were
improved but imp-6 and imp-7 were almost co eluted. To
improve the resolution between Imp-6 and Imp-7 buffer
pH adjusted to acidic side (0.01M NaH2PO4, pH 5.0),
(a)
(b)

120 P. SRINIVASU ET AL.
(c)
(d)
(e)

P. SRINIVASU ET AL.
121
(f)
(g)
(h)

P. SRINIVASU ET AL.
122
(i)
Figure 3. (a-e) Typical chromatograms from the method development trials; (f-i) Typical chromatograms of system suitability
and stressed fluocinonide samples.
Using the optimized conditions, fluocinonide, imp-1,
imp-2, imp-3, imp-4, imp-5, imp-6, imp-7 and imp-8
were well separated with a resolution of greater than 2
and typical retention times for imp-1, imp-2, imp-3,
imp-4, imp-5, fluocinonide, imp-6, imp-7 and imp-8, of
about 5.5, 9.2, 13.7, 14.6, 16.0, 21.8, 22.9, 24.4 and 24.9
min, respectively. The system suitability results are given
in Table 1 and the developed LC method was deter-
mined to be specific for fluocinonide and the eight impu-
rities, imp-1, imp-2, imp-3, imp-4, imp-5, imp-6, imp-7
and imp-8. (Table 2)
slight improvement in resolution between imp-6 and
imp-7 was observed (Rs < 1.0). To further improve the
resolution buffer pH was adjusted to 3.0 (0.01% phos-
phoric acid, pH 3.0). The resolution observed was 1.8.
Therefore, a pH value of 3.0 was selected for further
method development. Different gradient programs were
investigated and satisfactory results were obtained when
a gradient program of 0/40, 30/80, 35/80, 35.1/40 and
45/40 (time (min)/%B) was used.
Under the above described conditions, impurity spiked
solution and degradation samples were injected on a
cyano, phenyl, C8 and C18 columns having different
carbon loadings. On the cyano and phenyl columns,
imp-6 and imp-7 were co-eluted however, on C8 column
(Zorbax RX C8 with carbon loading ~8%), and C18
column (Agilent extend C18 column (carbon loading
~12%) the resolution between imp-6 and imp-7 was poor
(Rs < 1.2) .In contrast, when a Waters symmetry C18
250 mm x 4.6 mm, 5 µm column (carbon loading ~18%)
was used, satisfactory results were obtained. Based on
these experiments, the conditions were further optimized
as described below.
Table 1. System suitability report.
Compound USP Resolution
(RS)
USP Tailing
factor
No. of theoretical
plates
(USP tangent
method)
Imp-1 - 1.2 11248
Imp-2 14.9 1.2 20064
Imp-3 16.2 1.1 37955
Imp-4 3.2 1.1 44306
Imp-5 5.0 1.1 52387
Fluocinonide19.5 1.1 83484
Imp-6 3.6 1.1 87925
Imp-7 4.8 1.0 101784
Imp-8 2.1 1.0 99607
Waters symmetry C18 250 mm × 4.6 mm, 5µm column
was used as the stationary phase. The Mobile phase A
consisted of water, and its pH was adjusted to 3.0 using
phosphoric acid. The mobile phase B contained a mix-
ture of water and acetonitrile in the ratio of 150:850 (v/v).
The flow rate of the mobile phase was 1.0 ml/min with a
gradient program of 0/40, 30/80, 35/80, 35.1/40 and
45/40 (time (min)/%B). The column temperature was
maintained at 27°C and the detection was monitored at a
wavelength of 240 nm. The injection volume was 20 μl.

P. SRINIVASU ET AL.
123
Table 2. Summary of forced degradation results.
Stress condition Time % Assay of
active substance
Mass balance (%assay +
%impurities + % degra-
dation products)
Remarks
Acid hydrolysis
(1 M HCl) 48 h 86.5 99.4 One major degradation product was formed.
Base hydrolysis (0.1 M NaOH) 30 min 74.7 99.3 One major degradation product was formed.
Oxidation (5% H2O2) 48 h 90.8 99.3 One major degradation product was formed.
Thermal (60°C) 10 days 99.3 99.8 No degradation products formed
Light (photolytic degradation) 10 days 99.2 99.7 No degradation products formed
3.2. Method Validation
3.2.1. Precision
The %RSD of fluocinonide during the assay method pre-
cision study was within 0.5% and the %RSD values of
the area of imp-1, imp-2, imp-3, imp-4, imp-5, imp-6,
imp-7 and imp-8 in the related substance method preci-
sion study were within 1.0%. The %RSD of the results
obtained in the intermediate precision study was within
0.8% and the %RSD of the areas of imp-1, imp-2, imp-3,
imp-4, imp-5, imp-6, imp-7 and imp-8 were well within
1.8%, revealing the high precision of the method.
3.2.2. Limit of Detection and Limit of Quantification
The limits of detection and quantification of fluocinonide,
imp-1, imp-2, imp-3, imp-4, imp-5, imp-6, imp-7 and
imp-8 (analyte concentration of 250 g/ml) for a 20 l
injection volume are given in Table 3. The precision at
the LOQ concentration for imp-1, imp-2, imp-3, imp-4,
imp-5, imp-6, imp-7 and imp-8 was below 2.2%.
3.2.3. Linearity
The linear calibration plot for the assay method was ob-
tained over the tested calibration range (12.5-37.5 g/ml)
and the obtained correlation coefficient was greater than
0.999. The results revealed an excellent correlation be-
tween the peak area and analyte concentration. The slope
and y-intercept of the calibration curve for the ratio of
fluocinonide area and internal standard area were 0.0392
and 0.0286 respectively.
The linear calibration plot for the related substance
method was determined over the calibration ranges
(LOQ to 0.3%) for imp-1, imp-2, imp-3, imp-4, imp-5,
imp-6, imp-7 and imp-8, a correlation coefficient of
greater than 0.99 was obtained. The linearity was checked
for the related substance method over the same concen-
tration range for three consecutive days. The %RSD
values of the slope and y-intercept of the calibration
curves were 2.5 and 4, respectively. These results showed
an excellent correlation between the peak areas and con-
centrations of imp-1, imp-2, imp-3, imp-4, imp-5, imp-6,
imp-7 and imp-8.
3.2.4. Accuracy
The percentage recovery of fluocinonide in the drug sub-
stance and product ranged from 98.5 to 101.6 and from
96.2 to 101.6, respectively. The percentage recoveries of
imp-1, imp-2, imp-3, imp-4, imp-5, imp-6, imp-7 and
imp-8 in the drug substance and product ranged from
95.9 to 102.1 and from 96.3 to 102.5 respectively. The
HPLC chromatograms of spiked samples at the 0.15%
level of all four impurities in the fluocinonide drug sub-
stance sample are shown in Fi g ur e 3.
3.2.5. Robustness
In all of the deliberately varied chromatographic condi-
tions carried out as described in Subsection 2.3 (flow rate,
pH and column temperature), the resolution between the
any two peaks in system suitability solution was greater
than 2.0, illustrating the robustness of the method. The
assay variability of fluocinonide and the impurities was
within ± 1% and within ± 2%, respectively.
3.2.6. Solution Stability and Mobile Phase Stability
The %RSD of assaying fluocinonide during the solution
stability and mobile phase stability experiments was
within 1%. No significant changes were observed in the
content of Imp-1, imp-2, imp-3, imp-4, imp-5, imp-6,
imp-7 and imp-8 during the solution stability and mobile
phase stability experiments when performed using the
related substances method. The results of the solution
and mobile phase stability experiments confirm that the
sample solutions and mobile phase used during the as-
says and related substance determinations were stable up
to 48 h.
3.2.7. Results of Forced Degr adation Stu dies
Degradation was not observed in fluocinonide stressed

124 P. SRINIVASU ET AL.
Table 3. 1H-NMR and 13C-NMR assignments for fluocinonide and its degradation impurities.
Position Impurity at RRT 0.54 (Base Deg imp) Impurity at RRT 0.60 (Acid Deg imp)Fluocinonide
δ C δ H(J/HZ) δ C δ H(J/HZ) δ C δ H(J/HZ)
1 154.5 7.1(1H,d, 10) 155.4 7.15(1H,d,10) 155.9 7.2(1H,d,10)
2 128.8 6.3(1H,dd,10,1.5) 128.4 6.4(1H,dd,10,1.4) 127.8 6.4(1H,dd,10,1.4)
3 184.6 - 185.5 - 184.1 -
4 123.5 6.15(1H,s) 124.1 6.5(1H,s) 124.9 6.5(1H,s)
5 163.4 - 163.8 - 163.1 -
6 87.1 5.4(1H,dd) 87.5 5.4(1H,dd) 87.8 5.4(1H,dd)
7 34.2 1.58(2H,m) 33.7 1.58(2H,m) 34.4 1.58(2H,m)
8 31.5 1.68(1H,m) 32.3 1.68(1H,m) 32.3 1.67(1H,m)
9 101.5 - 100.7 - 100.9 -
10 47.4 - 47.9 - 47.9 -
11 70.1 4.45(1H,m) 70.9 4.45(1H,m) 70.1 4.45(1H,m)
12 33.9 1.63(2H,m) 33.5 1.63(2H,m) 33.6 1.63(2H,m)
13 36.1 - 36.6 - 36. 8 -
14 36.5 1.4(1H,m) 37.2 1.4(1H,m) 37.7 1.4(1H,m)
15 26.9 1.75(2H,m) 26 1.75(2H,m) 27 1.75(2H,m)
16 74.1 4.76(1H,m) 81.5 4.91(1H,m) 80.5 4.90(1H,m)
17 103.1 - 96.8 - 96.5 -
18 18.1 0.9(3H,s) 18.5 0.9(3H,s) 18.4 0.9(3H,s)
19 15.6 1.5(3H,s) 14.2 1.5(3H,s) 14.1 1.5(3H,s)
20 212.9 - 211.5 - 211.8 -
21 65.4 4.95(2H,s) 64.1 4.20,4.70(dd, 2H,19,6) 64.8 4.85(2H,s)
22 170.5 - 108.6 - 171.2 -
23 20.8 2.2(3H,s) 26.6 1.25(3H,s) 26.6 2.1(3H,s)
24 26.6 1.45(3H,s) 170.1 -
25 - - - - 26.2 1.20(3H,s)
26 - - - - 26.2 1.42(3H,s)
11-OH - 5.27(H,bd) - 5.29(1H,bd) - 5.25(1H,bd)
16-OH - 4.65(1H,d,6) - - -
17-OH - 4.85(1H,s) -
21-OH - - - 4.95(1H,t,6) - -

P. SRINIVASU ET AL.
125
samples subjected to light and heat. Significant degrada-
tion of the drug substance and product was detected un-
der acid, base hydrolysis and peroxide degradation,
leading to the formation of two major unknown degrada-
tion products one is at 0.54 RRT (due to base hydrolysis)
and another one is at 0.60 RRT (due to acid hydrolysis
and peroxide degradation) (Figure 1). Fluocinonide is
very sensitive towards base compared to acid and perox-
ide, within 30 minutes fluocinonide degraded 25% to an
unknown impurity at RRT 0.54. Peak purity test results
derived from the PDA detector, confirmed that the fluo-
cinonide peak and the degraded peaks were homogene-
ous and pure in all of the analyzed stress samples. Assay
studies were carried out for the stress samples against a
qualified reference standard of fluocinonide.
The mass balance of the stressed samples was close to
99.3%. The assay of fluocinonide was unaffected in the
presence of imp-1, imp-2, imp-3, imp-4, imp-5, imp-6,
imp-7, imp-8and its degradation products, confirming the
stability- indicating power of the developed method.
3.2.8. Identification of Major Degradation Products
Formed in Acid Hydrolysis, Peroxide
Degradation (RRT ~0.60) and Base Hydrolysis
(RRT ~0.54).
Degradation product in acid hydrolysis ( RRT ~0.60) and
base hydrolysis (RRT ~0.54) were isolated using a Shi-
madzu LC-8A preparative liquid chromatograph equipped
with SPD-10A VP, PDA detector (Shimadzu corporation,
Japan), Symmetry C18 (250 mm long × 19 mm i.d)
preparative column packed with 7 micron particle size
(Waters, USA). A mixture of water and acetonitrile in a
50:50 (v/v) ratio was used as the mobile phase. The flow
rate was 10 ml/min. The detection was carried out at 240
nm, Injection volume is 10 ml. Peak cut criteria was set
based on peak retention time. Fractions > 95% purity
were pooled together and concentrated by rotavapour to
remove solvents. Concentrated fraction was passed
through the preparative HPLC column again, the elute
was concentrated using rotavapour to remove solvents
then lyophilized using freeze dryer to obtain a white
powder with > 98% purity.
3.2.9. Structure Elucidation of Degradation Impurity
at RRT ~0.54
A LCMS study was carried to determine the m/z value of
the major degradation product formed under base hy-
drolysis using an Agilent 1100 series liquid chromatog-
raphy system coupled with a 6400 series triple quad-
rapole mass spectrometer. The volatile mobile phase
contained water, acetonitrile and acetic acid in a ratio of
60:40:1 (v/v/v) and the conditions were described in sec-
tion 2.4. The molecular ion peak m/z value obtained for
the degradation product resolving at 0.54 RRT is 455
[(MH)] +, in ESI positive mode corresponding to a mo-
lecular weights of 454.
In 1H-NMR and 13C-NMR spectra of new impurity
shows chemical shift similarity with fluocinonide, except
signals corresponding to C-16 and C-17. In 13C-NMR
C-16 and C-17 carbons resonated at δ 74.1 ppm and δ
103.1 ppm respectively (where as C-16 and C-17 carbons
in fluocinonide at δ 80.5 ppm and 96.5 ppm). In addition,
the hydroxyl groups on C-16 and C-17 resonated as dou-
blet at 4.65 ppm and as a singlet at 4.85 ppm respectively.
Comparative 1H-NMR and 13C-NMR spectral data for
fluocinonide and RRT ~0.54 is given in Table 3.
The IR Spectra performed on dispersion KBr of RRT
~0.54 degradant show the following absorption bands
3415, 3398 ~(broad, OH Stretching) when compared to
fluocinonide IR spectrum, a broad signal with a small
splitting(doublet) was observed in the region of 3200-
3500 which is a clear indication of 2 additional –OH
groups, 2950-2850 (CH,CH2 and CH3 Stretching) 1720
(C = O Saturated keto group), 1660 (C = O Conjugation)
1630 and 1610 (C = C Stretching)
The elemental analysis data calculated for C23H28F2O7
(454.46): C, 60.79; H, 6.21; F, 8.36; O, 24.64 and found:
C, 60.74; H, 6.18. From the above spectral data the deg-
radation impurity at RRT~0.54 was confirmed as
pregna-1, 4-diene-3, 20-dione, 21-(acetyloxy)-6, 9-di-
fluoro-11, 16, 17-trihydroxy-(6α, 11β, 16α)-.
3.2.10. Structure Elucidation of Degradation
Impurity at RRT ~0.60
A LCMS study was carried to determine the m/z value of
the major degradation product formed under acid hy-
drolysis using an Agilent 1100 series liquid chromatog-
raphy system coupled with a 6400 series triple quad-
rapole mass spectrometer. The volatile mobile phase
contained water, acetonitrile and acetic acid in a ratio of
60:40:1 (v/v/v) and the conditions were described in sec-
tion 2.4. The molecular ion peak m/z value obtained for
the degradation product resolving at 0.60 RRT is 454
[(MH)]+, in ESI positive mode corresponding to a mo-
lecular weights of 452.5.
In 1H-NMR and 13C-NMR spectra of new impurity
shows chemical shift similarity with fluocinonide, except
signals corresponding to C-21. In 1H-NMR the two pro-
tons on C-21 resonated as doublet of doublet at 4.20 &
4.70 ppm (J = 19 and 6 HZ) and the corresponding hy-
droxyl proton as triplet (which is a double doublet and
appearing as triplet because of masking effect) at 4.95
ppm. Comparative 1H-NMR and 13C-NMR spectral data
for fluocinonide and RRT ~0.60 is given in Table 3.
The IR Spectra performed on dispersion KBr of RRT
~0.60 degradant show the following absorption bands

126 P. SRINIVASU ET AL.
~3500 (broad OH Stretching), 2950-2850 (CH, CH2 and
CH3 Stretching) 1720 (C = O Saturated keto group), 1660
(C = O Conjugation) 1630 and 1610 (C = C Stretching)
The elemental analysis data calculated for C24H30F2O6
(452.49): C, 63.70; H, 6.68; F, 8.4; O, 21.22 and found:
C, 63.74; H, 6.64. From the above spectral data the
degradation impurity at RRT ~0.60 was confirmed as
pregna-1, 4-diene-3, 20-dione, 6, 9-difluoro-11, 21 dihy-
droxy-16, 17-[(1-methylethylidene) bis(oxy)]-, (6α, 11β,
16α)-.
4. Conclusions
In this paper, a sensitive, specific, accurate, validated and
well-defined stability indicating LC method for the de-
termination of fluocinonide in the presence of degrada-
tion products and its process-related impurities was de-
scribed. The behavior of fluocinonide under various
stress conditions was studied, and the hydrolysis (acid
and base) degradants were identified by LCMS and other
spectral analysis presented. All of the degradation prod-
ucts and process impurities were well separated from the
drug substance and drug product demonstrates the stabil-
ity-indicating power of the method. The information
presented in this study could be very useful for quality
monitoring of drug substance and its dosage forms and
be used to check drug quality during stability studies.
5. Acknowledgements
The authors wish to thank the management of Ver-
sapharm Incorporated for supporting this work. They
also thank Mr. Naresh Chintalapati, Mr. Ramalingaraju
Gadiraju, Dr. Ramanujachary Kandalam, Dr. Vijay Krishna
Kari, Dr. Rajendra Prasad Kalakodimi and Dr. Sivaku-
mar Vasireddy for their technical support in carrying out
this work.
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