INTRODUCTION:

Fluocinolone acetonide (FA), 6α, 9α-diFAoro-11β, 21-dihydroxy-16α, 17α-isopropylidenedioxy-1, 4-diene, 3, 20-Dione (fig. 1) is a corticosteroid used topically for treatment of a variety of skin disorders and inflammatory eye, ear, and nose diseases ^[¹^]. It has high anti-inflammatory activity and is usually used formulated as a cream, gel, lotion, or ointment ^[²^,³^]. Miconazole nitrate (MCZ), 1H-imidazole, 1-[2-(2, 4-dichlorophenyl)-2-[(2, 4- icholophenyl) methoxy] ethyl] - (±) nitrate (Fig. 1) is an antimycotic imidazole derivative^[¹^]^. With a wide activity spectrum, it is endowed with a powerful activity against dermatophytes and Candida albicans, as well as against several Gram positive germens ^[³^,⁴^]^.

The generally accepted mode of action of azole antifungals is the inhibition of 14-alpha-lanosterol demethylase, a key enzyme in ergosterol biosynthesis, resulting in depletion of ergosterol and accumulation of toxic 14-alpha-methylated sterols in the membrane of susceptible yeast species^[⁵^]MCZ has been extensively applied in the management of dermal, oral, and vaginal mycosis. Currently, it is used in a variety of pharmaceutical formulations such as injections, tablets, oral gels, creams, ointments, topical powders and vaginal suppositories. The most usual application forms include creams, ointments or gels at 2.0% concentration level, alone or associated with anti-inflammatory steroids, or other antimicrobials such as gentamycin for the treatment of dermatitis ^[⁶^,⁷^].

(a)FA

(b) MCZ

Figure: 1 Chemical structures of (a) FA and (b) MCZ

Literature review reveals that detection and quantitative determination of FA and MCZ in routine analysis of individual drug, with combination of other drugs as well as in biological matrices, including human plasma, serum, and urine, is accomplished by variety of methods, including potentiometry^[¹^,⁸^], spectrophotometry^[⁹^,10], high performance liquid chromatography (HPLC)^[10-21], HPLC with spectrophotometry^[¹⁴^,18], HPLC with mass spectroscopy (LC-MS)^[22-24], gas chromatography (GC)^[⁸^,25], thin layer chromatography (TLC)^[26], high performance thin layer chromatography (HPTLC)^[27], etc in various dosage forms like cream, gel, tablets, ovule, ointment. However best of our knowledge, no method has been reported yet for simultaneous estimation of above mentioned drugs.

The International Conference on Harmonization (ICH) guideline entitled Stability Testing of New Drug Substances and Products requires the stress testing to be carried out to elucidate the inherent stability characteristics of the active substance ^[²⁸^]. Susceptibility to oxidation is one of the required tests. Also the hydrolytic and the photolytic stability are required. An ideal stability-indicating method is one that quantifies the standard drug alone and also resolves its degradation products. Ferenczi-Fodor and colleagues^[²⁹^]explained basic acceptance criteria for evaluation of validation experiments based on practical experience for planar chromatographic procedures, which may be used at different levels either in qualitative identity testing, assays, semi-quantitative limit tests or quantitative determination of impurities. The parameters for robustness testing of the given procedures and quality assurance of quantitative planar chromatographic testing have been described as per ICH guidelines.

Nowadays HPTLC is becoming a routine analytical technique due to its advantages of low operating cost, high sample throughput and need for minimum sample clean up. The major advantage of HPTLC is that several samples can be run simultaneously using a small quantity of mobile phase unlike HPLC, thus lowering the analysis time and cost peranalysis.

The aim of the present work was to develop and validate a new simple, rapid, selective, cost effective and stability indicating HPTLC method for simultaneous determination of FA and MCZ in ointment formulation in the presence of its degradation products. The proposed method was validated as per ICH guidelines ^[³⁰^,³¹^].

EXPERIMENTAL:

Materials

Pharmaceutical grade of MCZ (Luxica Pharma Inn., Ankleshwer, Gujarat, India) and FA (Dermocare Labs Ltd., Ahmedabad, Gujarat, India) were kindly supplied as a gift sample. All chemicals and reagents used were of analytical grade and were purchased from Merck Chemicals, India.

Chromatographic Conditions

The samples were spotted in the form of bands of width6mmwith a Camag 100 µL syringe on pre-coated silica gel aluminium Plate 60 F–254 (20 × 10 cm with 250 µm thickness; E. Merck, Darmstadt, Germany, supplied by Anchrom Technologists, Mumbai, India) using a Camag Linomat V (Muttenz, Switzerland). The plates were prewashed by methanol and activated at 60 °C for 5 min prior to chromatography. A constant application rate of 0.1µl/s was employed and space between two bands was 5 mm. The slit dimension was kept at 5 × 0.45 mm and scanning speed was employed 10 mm/s. The monochromatic bandwidth was set at 20 nm, each track was scanned thrice and base line correction was used. The mobile phase consisted of n-Hexane/Ethyl acetate (1:9 v/v) and 10 ml of mobile phase was used as per chromatography. Linear ascending development was carried out in 10 × 10 cm twin trough glass chamber (Camag, Muttenz, Switzerland) saturated with the mobile phase. The optimized chamber saturation time for mobile phase was 30 min at room temperature (25 °C ± 2) at relative humidity of 60 ± 5%. The length of chromatogram run was 8 cm.Subsequent to the development; TLC plates were dried in a current of air with the help of an air dryer. Densitometric scanning was performed on Camag TLC scanner III in the reflectance–absorbance mode at 260 nm and was operated by CATS software (V 3.15, Camag). The source of radiation utilized was deuterium lamp emitting a continuous UV spectrum between 190 and 400 nm. Concentrations of the compound chromatographed were determined from the intensity of diffusely reflected light. Evaluation was via peak areas with linear regression.

Preparation of standard solutions

Accurately weighed portion of MCZ (250 mg) and FA (25 mg) was transferred to a separate 25 ml volumetric flask and dissolved and diluted to the mark with methanol to obtain standard solution having concentrations of MCZ (10000µg/ml) and FA (1000µg/ml). Further dilute FA 10 times to obtain standard stock solution having concentration 100 µg/ml.

Preparation of sample solutions:

Weigh an accurately about 2 grams of ointment containing 2% of MCZ and 0.01% of FA in 10 ml volumetric flask. Add 2/3rd quantity of methanol into flask and sonicate the solution for 10 min to get clear solution. Add methanol up to mark to make the volume 10 ml. get the test solution contains 4000µg/ml MCZ and 20 µg/ml FA.

Preparation of calibration curve

To set up the linearity range 40000-140000 ng/spot for MCZ and 200-700 ng/spot FA. From the combined working standard solution (20 μg/ml of FA and 4000 μg/ml of MCZ) aliquots of 10, 15, 20, 25, 30 and 35 μl were spotted on the TLC plate and developed and analysed. The above working standard of MCZ and FA were spotted in band width 6 mm using Hamilton 100 µL syringe on pre-coated silica gel aluminium plate 60 F₂₅₄using automatic application device. Linear ascending development was carried out in 10 × 10 cm twin trough glass chamber saturated with the mobile phase for 30 min. The plate was removed from the chamber, subsequently dried in a current of air and densitometric scanning was performed on Camag TLC scanner III in the reflectance-absorption mode at 254 nm and operated by win CATS software. Peak areas were recorded for all the peaks. The calibration curve for MCZ and FA were constructed by plotting peak area versus concentration (ng/spot) corresponding to each spot.

Method validation

Precision

Repeatability

System precision experiment was performed by application of 15 µL of combined working standard solution (20 µg/mL of FA and 4000 µg/mL of MCZ) for six times on same TLC plate. Plate was developed and analyzed as described in section 4.2.1. The areas of six replicate spots were measured and % RSD was calculated.

Intermediate Precision (Reproducibility)

The intra-day and inter-day precision of the proposed method was determined by analyzing mixed standard solution of FA and MCZ at 3 different concentrations (200, 400, 600 ng/spot for FA and 40000, 80000, 120000 ng/spot for MCZ) on the same day and on different days. The results are reported in terms of relative standard deviation (%RSD).

Robustness of the method

By introducing small changes in the mobile phase composition, the effects on the results were examined. Mobile phases having different composition like n- Hexane/ Ethyl acetate (0.9:9.1 v/v) and (1.1:8.9, v/v) were tried and chromatograms were run. The amount of mobile phase, temperature and relative humidity was varied in the range of ± 5%. The plates were prewashed by methanol and activated at 60±5°C for 2,5and7min. prior to chromatography. Time from spotting to chromatography and from chromatography to scanning varied from 0,25 and 35 min. Robustness of the method was done at4000 ng/spot of MCZ and 20 ng/spot FA.

Limit of detection and limit of quantitation

The limits of detection and quantification of the developed method were calculated from the standard deviation of the intercepts and mean slope of the calibration curves of FA and MCZ using the formulae as given below.

LOD = 3.3 Χσ/S

LOQ = 10 Χσ/S

Where, σ = the standard deviation of the response

S = slope of the calibration curve.

Specificity

The specificity of the method was ascertained by analysing standard drug and sample. The spot for MCZ and FA in sample was confirmed by comparing the Rf and spectra of the spot with that of standard. The peak purity of MCZ and FA were assessed by comparing the spectra at three different levels; i.e. peak start (S), peak apex (M) and peak end (E) positions of the spot.

Accuracy

The samples were spiked with extra 80%, 100%and 120% of the standard MCZ and FA solutions, and the mixtures were reanalysed by the proposed method. The experiment was conducted in triplicate. This was done to check for the recovery of the drug at different levels in the formulations.

Analysis of the marketed formulation

20 µl of sample solution was applied on the TLC plate followed by development and scanning as described in Section 2.2. The analysis was repeated in triplicate. The amount MCZ and FA present in the sample solutions were determined by fitting the response into the respective regression line equation for MCZ and FA.

Forced degradation

Forced degradation of FA and MCZ was carried out under acidic and alkaline hydrolysis, oxidative and photolytic stress conditions.

Acidic hydrolysis

Accurately weighed 2 mg of FA and 400 mg of MCZ was transferred into 10 ml volumetric flask, dissolved in 5 ml methanol and diluted up to the mark with 2N HCl. The solution was refluxed for 1 hour at room temperature. Aliquot of 1 ml was transferred to 10 ml volumetric flask and neutralized by 2N NaOH. Volume was made up to mark with methanol. The resulting solution (15 µl) was applied to TLC plate and the chromatogram was developed.

Alkaline hydrolysis

Accurately weighed 2 mg of FA and 400 mg of MCZ was transferred into 10 ml volumetric flask, dissolved in 5 ml methanol and diluted up to the mark with 2N NaOH. The solution was refluxed for 1 hour at room temperature. Aliquot of 1 ml was transferred to 10 ml volumetric flask and neutralized by 2N HCl. Volume was made up to mark with methanol. The resulting solution (15 µl) was applied to TLC plate and the chromatogram was developed.

Oxidative degradation

Accurately weighed 2 mg of FA and 400 mg of MCZ was transferred into 10 ml volumetric flask, dissolved in 5 ml methanol and diluted up to mark with 6% hydrogen peroxide (H2O2). The solution was kept at room temperature (25± 20C) for 1 hour. From sample solution 1 ml aliquot was transferred in to 10 ml volumetric flask and volume made up to mark with methanol. The resulting solution (15 µl) was applied to TLC plate and the chromatogram was developed.

Photolytic degradation

For photolytic degradation studies, working standard solution of FA (20µg/ml) and MCZ (4000µg/ml) was exposed to direct sunlight (afternoon, 32±0.5°C) for 3 hr on a wooden plank kept on terrace. 15 µl of the solution was applied on TLC plate and the chromatogram was developed.

RESULT AND DISCUSSION

Mobile phase optimization

The HPTLC procedure was optimized with a view to develop stability indicating assay method. Both, MCZ and FA were spotted on TLC plates and run in different mobile phase systems. The mixture of n-Hexane: Ethyl acetate (1:9, v /v) was proven to be better than the other mixtures in terms of resolution and peak shape.

Validation of the proposed method

Specificity

The peak purity of FA and MCZ were assessed by comparing its HPTLC chromatogram with standard at peak start, apex, and peak end positions of the spot.

Good correlation (r= 0.9990) was obtained between standard and sample spectra of FA and MCZ respectively.

Peak purity spectra of test FA and MCZ are shown in Figure: 2 and 3 respectively. Data of peak purity are shown in Table 1.

Table 2: Calibration data of MCZ

Sr. No.	Concentration (ng/spot)	Peak Area ± S.D. (n= 6)	%RSD
1	40000	7485.3±93.2	1.24
2	60000	10019.4±91.2	0.91
3	80000	12737.1±94.9	0.74
4	100000	14562±271.07	1.86
5	120000	16625.3±266.86	1.60
6	140000	20387±259.83	1.27

Table 3: Calibration data of FA

Sr. No.	Concentration (ng/spot)	Peak Area ± S.D. (n= 6)	%RSD
1	200	1466.9±26.55	1.81
2	300	2191.6±10.95	0.49
3	400	2701.6±21.54	0.79
4	500	3202.3±32.50	1.01
5	600	3441.4±32.27	0.93
6	700	4161.7±22.30	0.53

Precision

Ø System and method precision

%RSD of system precision of FA and MCZ were found to be 1.37 and 1.59 respectively. %RSD of method precision of FA and MCZ were found to be 1.75 and 1.63 respectively. The data for system and method precision of FA and MCZ are depicted in Table 4 and 5 respectively.

Ø Intra-day and Inter-day precision

Mean RSD for intra-day and inter-day precision of MCZ was found to be 1.36 and 1.32 respectively. The Mean RSD for interaday and inter day precision of FA was found to be 0.6818 and 1.36 respectively. The data for intra-day and inter-day precision for MCZ is depicted in Table 6 and for FA in Table 7. These results indicate that method is precise.

Table 5: Intermediate precision data for MCZ

Concentration of MCZ (ng/spot)	Intra-day precision		Inter-day precision
Concentration of MCZ (ng/spot)	Area Mean ± SD(n= 3)	%RSD	Area Mean ± SD(n= 3)	%RSD
60000	10019.4 ± 91.20	0.9102	10453.67 ± 146.79	1.40
100000	14562.267 ± 271.07	1.86	14453.67 ± 146.791	1.0155
140000	20387 ± 270.687	1.31	20953.67 ± 322.108	1.548
Mean RSD		1.36	Mean RSD	1.32

Table 6: Intermediate precision data for FA

Concentration of FA (ng/spot)	Intra-day precision		Inter-day precision
Concentration of FA (ng/spot)	Area Mean ± SD (n= 3)	%RSD	Area Mean ± SD (n= 3)	%RSD
300	2191.6 ± 10.95	0.50	2191.6 ± 10.95	0.85
500	3202.3 ± 32.50	1.01	3202.3 ± 32.50	1.60
700	4161.7 ± 22.3	0.53	4161.7 ± 22.3	1.65
Mean RSD		0.68%	Mean RSD	1.36%

Table 7: Recovery data of FA from formulation

Sr. No.	Amount taken (ng/spot)	Amount added (ng/spot)	Total (ng/spot)	Area	Amount Recover (ng/spot)	% Recovery	Mean % Recovery
1	300	240	540	3320.6	241.04	100.43	100.63
		240	540	3293.6	235.71	98.21
		240	540	3354.9	247.81	103.25
2	300	300	600	3621.4	300.39	100.13	99.44
		300	600	3615.7	299.27	99.75
		300	600	3595.6	295.30	98.43
3	300	360	660	3933.4	361.95	100.54	100.41
		360	660	3914.6	358.24	99.51
		360	660	3945.2	364.28	101.19
Mean Recovery = 100.16%
Standard Deviation = 0.63
% Relative Standard Deviation = 0.63%

Table 8: Recovery data of MCZ from formulation

Sr No.	Amount taken (ng/spot)	Amount added (ng/spot)	Total (ng/spot)	Area	Amount Recover (ng/spot)	% Recovery	Mean % Recovery
1	60000	48000	108000	15956.4	48918.71	101.91	99.59
		48000	108000	15865.4	48182.13	100.38
		48000	108000	15635.7	46314.65	96.49
2	60000	60000	120000	17213.6	59143.14	98.57	98.73
		60000	120000	17358.4	60320.36	100.53
		60000	120000	17105.9	58267.51	97.11
3	60000	72000	132000	18667.4	70962.65	98.55	97.88
		72000	132000	18420.9	68958.53	95.77
		72000	132000	18735.1	71513.11	99.32
Mean Recovery = 98.73%
Standard Deviation = 0.85
% Relative Standard Deviation = 0.86%

Table 9:LOD and LOQ data for FA and MCZ nitrate

Parameters	FA	MCZ
Standard deviation of the Y- intercepts of the three calibration curves.	17.71	107.33
Mean slope of the three calibration curves.	5.06	0.12
LOD = 3.3 × (SD/Slope) (ng/spot)	11.54	2887.56
LOQ = 10 × (SD/Slope) (ng/spot)	34.97	8750.20

Table 10: Robustness parameters for FA and MCZ

Sr. No.	FA		MCZ
Change in saturation time
	25 min	35 min		25 min
1	2114.7	2123.1	1	2114.7
2	2126.7	2107.5	2	2126.7
Mean	2118		9955.7
S.D	8.61		109.38
%RSD	0.40		1.09
Chang in Mobile phase ratio: n- Hexane: Ethyl acetate
	0.9:9.1, v/v	1.1:8.9, v/v		0.9:9.1, v/v
1	2058.28	2060.63	1	2058.28
2	2112.51	2131.08	2	2112.51
Mean	2102.6		9962.63
S.D	26.06		89.92
% RSD	1.23		0.90
Change in sonication time for sample preparation:
	5 min	15 min		5 min
1	2102.7	2113.1	1	2102.7
2	2085.4	2107.5	2	2085.4
Mean	2102.175		10090.62
S.D	11.96		120.17
% RSD	0.56		1.19

Table 13: Integration parameters for HPTLC method

Parameters	Value
Video integration	no
Baseline correction	yes
Peak thresh. height	10 AU
Peak thresh. area	300
Peak thresh. slope	3
Filter factor	3
Data selection factor	1
Track start position	85 mm
Track end position	135 mm

Table 14: Peak table of acid degradation study

Peak No.	R_f value	% Area	Substance
1	0.14	10.65	Degradation
2	0.32	14.79	Degradation
3	0.50	49.47	MCZ
4	0.64	19.53	FA
5	0.87	5.26	Degradation

Alkaline hydrolysis

The drug was found to be highly labile to alkaline hydrolysis. After applying 1 N NaOH exposure for 1 hour, about 40% degradation was observed. Additional peak of degradation product were observed at R_f 0.36, 0.42 and 0.59 (Figure 9). Data of alkaline hydrolysis is given in table 15.

Table 15: Peak table of alkali degradation

Peak No.	R_f value	% Area	Substance
1	0.36	12.71	Degradation
2	0.42	10.65	Degradation
3	0.51	44.62	MCZ
4	0.59	16.53	Degradation
5	0.64	15.49	FA

Oxidative degradation

The drugs were found to be slightly labile to oxidative degradation. After applying 3% hydrogen peroxide (H₂O₂) at room temperature (25 ± 2⁰C) for one hour, about 16.17% degradation was observed. Additional two degradation peaks are observed at R_f 0.21 and 0.29 (Figure 10). Data of oxidative degradation is given in table 16.

Figure 11: Densitogram of photodegradation

Table 17: Peak table of Photo degradation

Peak No.	R_f value	% Area	Substance
1	0.23	8.76	Degradation
2	0.39	9.39	Degradation
3	0.46	47.88	MCZ
4	0.54	17.48	Degradation
5	0.66	16.49	FA

Table 18: Summary of forced degradation study of FA and MCZ

Sr. No.	Stress type*	Stress condition	Peak area of FA	Peak area of MCZ	% Degradation
1	Acid hydrolysis	1 N HCl at Room temperature, for 1 hour	973.1	4153.8	30.7%
2	Alkali hydrolysis	1 N NaOH at Room temperature, for 1 hour	1837.1	5374	40%
3	Oxidation	3% W/V H₂O₂ at Room temperature, for 1 hour	2430	10514	16.17%
4	Photolytic degradation	Direct sunlight for 3 hour	976.8	12018.9	35.63%

*Concentration of FA: 300 ng/spot and for MCZ: 60000 ng/spot

CONCLUSION:

Based on the results, obtained from the analysis using described method, it can be concluded that the method has linear response in the range of 200-700 ng/spot for FA and 40000 – 140000 for MCZ.

The result of the analysis of pharmaceutical formulation by the proposed method is highly reproducible and reliable and is in good agreement with label claim of the drugs. The additive usually present in the pharmaceutical formulations of the assayed samples did not interfere with MCZ and FA. The method can be used for the routine analysmethod successfully separated drug substances from degradation products formed under various stress conditions. MCZ and FA are prone to alkaline hydrolysis and photolysis.

ACKNOWLEDGEMENT:

The authors are thankful to Luxica Pharma Inn. and Dermocare Labs Ltd. for MCZ and FA as gift samples.

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Received on 06.04.2015 Accepted on 28.05.2015

Asian J. Pharm. Ana. 5(2): April-June 2015; Page 49-60

DOI: 10.5958/2231-5675.2015.00009.5

Substance	r(s, m)	r(m, e)
Fluocinolone acetonide	0.9996	0.9995
Miconazole nitrate	0.9999	1.000

Sr.No.	Peak Area
Sr.No.	MCZ(60000 ng/spot)	FA(300 ng/spot)
1	9828.4	2114.7
2	10069.7	2126.7
3	10019.8	2123.1
4	9904.9	2107.5
5	10184.6	2102.4
6	10143.2	2195
Mean	10025.19	2128.23
S.D.	137.51	33.96
% RSD	1.37	1.59

Drug	Label claim	Amount found	% Content
FA	0.01% w/w	0.009882% w/w	98.82%
MCZ	2.0% w/w	1.9994% w/w	99.97%

Sr. No.	Parameters	FA	MCZ
1	Linearity Range	200-700 ng/spot	4000-140000 ng/spot
2	Correlation Co-efficient (R²)	0.990	0.983
3	Precision (% RSD) Repeatability (n=6) Intraday precision (n=3) Inter day precision (n=3)	1.54 0.681 1.366	1.76 1.36 1.32
4	% Recovery	100.163%	98.79%
5	Limit of Detection (LOD)	11.5424	2887.56
6	Limit of Quantification (LOQ)	34.9769	8750.20

Accuracy

Limit of Detection (LOD) and Limit of Quantification (LOQ)

Analysis of marketed formulations (ointment) by proposed method

Stability indicating property

Acid hydrolysis

Alkaline hydrolysis