INTRODUCTION:

Terconazole (TER) (Fig. 1) is an anti-fungal medication, primarily used to treat vaginal fungal infections.TER is chemically1-(4-{[(2R,4S)-2-(2,4-dichlorophenyl)-2-(1H-1,2,4-triazol-1-ylmethyl)-1,3-dioxolan-4-yl]methoxy}phenyl)-4-(propan-2-yl)piperazine^1,
2.TER is a triazole antifungal agent available for intravaginal use. It is structurally related to imidazole-derivative antifungal agents, although TER and other triazoles have three nitrogens in the azole ring. TER may exert its antifungal activity by disrupting normal fungal cell membrane permeability. TER and other triazole antifungal agents inhibit cytochrome P450 14-alpha-demethylase in susceptible fungi, which leads to the accumulation of lanosterol and other methylated sterols and a decrease in ergosterol concentration. Depletion of ergosterol in the membrane disrupts the structure and function of the fungal cell leading to a decrease or inhibition of fungal growth^3-5.

Figure 1- Structure of TER

Literature survey revealed that there is very limited number of methods available for estimation of TER. Instead, the analytical methods developed for other drugs of same category utilized in which TER used as internal standard and provide some hint for method development of TER^6-8.The analytical methods developed for TER includes the optimization of different experimental conditions in spectroscopy for the determination of TER in the Gyno-fungix cream⁹, spectrophotometric determination of poorly water soluble drug TER using hydrotropic solubilization technique¹⁰and UV-spectrophotometric method for the estimation of TER in Bulk and Pharmaceutical Dosage Form¹¹.

The present work is based on the development of High Performance Thin layer chromatography tandem mass spectrometry (HPTLC-MS).Thin layer chromatography (TLC) is a simple, cost-effective and easy chromatographic technique that has been used in identification and quantification of various drugs as well as biological samples of interest. Traditionally, chemical and optical methods are employed to visualize the analyte spots on the TLC plate. Because direct identification and structural characterization of the analytes on the TLC plate through these methods are not possible effectively. But, it seems very convenient to combine TLC with mass spectrometry (MS) which is one of the most efficient analytical tool for structural elucidation¹².This method is very rapid as well selective as compared to other sophisticated techniques.

MATERIALS AND METHODS:

Apparatus: Camag HPTLC system comprising of Camag Linomat V semiautomatic sample applicator, Camag TLC Scanner 3, Camag twin-trough developing chamber (10 X 10 cm), UV cabinet, Camag Win-Cat software, Hamilton syringe (100 µl), API 4000 Q TRAP (ABSCIEX, CA, USA) LC-MS/MS spectrometer fitted with an electro spray ionization interface and analytical balance were used in the present research work.

Reagents and Materials:

Silica Gel 60 F₂₅₄ TLC plates (10 X 10 cm, layer thickness 0.2 mm, E. Merck, Darmstadt, Germany) were used as stationary phase. Methanol, Ethyl acetate and Toluene (AR grade, Fisher Scientific, India) were used for mobile phase preparation.

Preparation of standard and sample solution:

10 mg of TER was weighed accurately, transferred to 10 ml volumetric flask and diluted to 10 ml using methanol to get concentration 1 mg/ml. From this stock solution, 0.5 ml was further diluted to 10 ml using methanol to get solution having concentration 50ng/µl.

Preparation of matrix for analysis:

For the analysis of TER in pharmaceutical formulation, matrix was prepared using selected excipients along with biological components according to formula noted in Table No. 1.

Table No.1- Master Formula for biological matrix

Constituents	Amount (g)
Glucose	2.2065
Lactic Acid	0.5
Acetic Acid	0.013
Albumin	0.2164
Urea	1.0432
NaCl	0.2621
KCL	0.2485
Ca Chloride	0.026
Glycerol	0.1
Distilled water	up to 250 ml.

Chromatographic conditions:

The experiment was performed on silica gel 60 F₂₅₄ aluminum sheets (10 X 10 cm) as stationary phase, using mobile phase comprised of Toluene: Ethyl Acetate: Methanol (6:2:2 v/v/v). TLC plates were prewashed with methanol and activated in an oven at 110ºC for 10 min prior to chromatographic experiment. The solutions were applied on TLC plate in the form of bands of 6 mm width under a stream of nitrogen gas using a Camag Linomat V semiautomatic sample applicator. Ascending development to 70 mm was performed in 10 cm x 10 cm Camag twin trough glass chamber saturated with the mobile phase for 15 min. The developed TLC plate was air dried and then scanned between 200 to 400 nm using Camag TLC scanner 3 using Win Cat software. TER showed reasonably good response at 230 nm keeping the slit dimension of 6.00 x 0.30 mm and scanning speed of 20 mm/s (Fig 2).

Figure 2-In situ spectrum of TER measured from 200 to 400 nm.

Summary of Chromatographic Parameters Selected:

a	Solvent used	Methanol
b	Stationary phase	TLC plate precoated with silica gel 60 F₂₅₄
c	Mobile phase	Toluene: Ethyl Acetate: Methanol (6: 2: 2 v/v/v)
d	Chamber saturation time	15 min
e	Development time	20 min
f	Detection wavelength	230
g	Temperature	Ambient

Linearity and calibration graph:

Linearity was obtained over the concentration range 50 - 300 ng/band by applying 1 – 6 µl from standard solution (50ng/µl) as a band of 6mm width on the TLC plate. All the chromatographic conditions were maintained as above. After completion of chromatographic analysis, TER showed Rf value0.65±0.018 along with Asymmetry factor 1.1 (Fig.3).Peak areas of TER were plotted against corresponding concentrations and least square regression analysis was performed to generate the calibration equation (Fig 4).

Figure 3-Densitogram of standard TER (100 ng/band, R_f = 0.65 ± 0.018)

Figure 4-Calibration graph of TER

Application of the proposed method for estimation of TER in biological matrix:

Appropriate volume 2 ml from stock solution (1 mg/ml) was withdrawn in 10 ml volumetric flask and volume was adjusted up to mark with methanol. This standard solution having concentration 200µg/ml was used for estimation of TER in biological matrix.

Spiked samples (50ng/μl, 6 replicate) were prepared by adding 0.25 ml solution from above standard solution of TER (400 µg/ml) to 0.25 ml of prepared biological matrix and making volume to 1 ml with methanol. The contents of the tubes were vortexed for 3 min and then centrifuged for 10 minutes at 2500 rpm. After centrifugation, 2 µl supernatant aliquots of each replicate were applied as bands on the plate and the plate was developed as per the standard conditions. Results are shown in Table No.2.

Table No.2-Estimation of TER in biological matrix

Concentration

(ng/band)

Area (µV. Sec) (Average, n = 6)

Mean % Recovery* ± SD

% RSD*

100

2257.127

98.07 ± 1.2

1.255

* Average of six determinations

(ng-nanogram, n- number of readings, SD- standard deviation, RSD- Relative standard deviation)

HPTLC-MS conditions:

The triple quadrupole system was an API 4000 Q TRAP (ABSCIEX, CA, USA) LC-MS/MS spectrometer fitted with an electro spray ionization interface. The ESI-MS was operated in both positive and negative detection mode. Calibration of the mass analyzer was performed by infusion (10µl min^-1) of a commercial mixture of polypropylene glycol (PPG) which was supplied by AB SCIEX using a 1ml Hamilton syringe and monitored eight mass-to-charge ratios (m/z) in the 59-1800 mass range. The ESI source conditions were: ion spray voltage, 5500 V; nebulizer gas (GS1), 50 psi; curtain gas, 25 psi; turbo gas (GS2), 50 psi; collision gas (CAD), 7 psi and ion source temperature 470⁰ C. The pesticides detection and quantification were performed in schedule multi reaction mode (schedule MRM). Q1 and Q3 quadrupoles set to unit resolution. Data acquisition and quantification were carried out using Analyst software version 1.5. The mass spectrum of TER was displayed as reported in Fig. 5.

Figure 5- Full scan mass spectrum of TER

VALIDATION OF HPTLC METHOD:

Validation of the proposed method was carried out with various parameters such as precision, accuracy, specificity, LOD, LOQ

Accuracy and Precision:

Intra and inter day accuracy and precision evaluations were performed by repeated analysis of TER in biological matrix. The run consisted of a three replicates of each 100, 150, 200 ng/band of TER. The overall precision of the method expressed as relative standard deviation and recovery of the method. Intraday accuracy ranged from 99.37 % to 98.52 % and precision was 0.51. Inter day accuracy ranged from 98.33% to 100.68% and precision was 1.19. The mean Recovery, standard deviation (SD), coefficient of variation (% RSD) was evaluated and their results were tabulated in Table No. 3.

1. Specificity:

Specificity is the ability to measure accurately and specifically the analyte of interest in the presence of other components that may be expected to be present in the sample matrix. The specificity of the proposed method is illustrated in Fig. 6 where a blank sample of matrix was spotted on the plate. The densitogram showed that there were no endogenous peaks at the retention time of TER.The components of biological matrix did not interfere with the analyte, thereby confirming the specificity of the analytical method.

Figure 6- Densitogram of Blank sample of matrix

2. LOD and LOQ:

The limit of Detection and limit of Quantitation were calculated for TER by using the standard deviation and slope obtained from linearity studies and it was found to be LOD 11.5ng / band and LOQ 34.9ng / band.

RESULTS AND DISCUSSIONS:

The reported method is based on the principle of chromatographic separation and spectrometric identification of TER in the biological matrix. HPTLC and MS methods were optimized separately by number of trials and finally specifications were decided which are enlisted in table.TER showed Rf value 0.65±0.018 along with Asymmetry factor 1.1 using Toluene: Ethyl Acetate: Methanol (6:2:2 v/v/v) as mobile phase. Linearity was obtained within concentration range 50-300ng/band with regression coefficient 0.9921. Sample recovery was found to be 98.1±1.2% when applied to biological matrix. Mass spectrometry resulted in the spectra showing base peak at531.8 indicating mass of molecular ion of TER. Similarly, mass peaks were observed at 125.6, 177.3, 192.3, 213.3, 234.2, 247.4, 255.8, 277.3, 312.2 and 489.8 indicating other fragments of TER.

The developed method was validated by performing various parameters in which the accuracy and precision was evaluated based on percentage recovery and percentage relative standard deviation. The mean percentage recovery was found to be 99.1±0.51with mean % RSD 0.512 for intraday accuracy and precision. Similarly mean percentage recovery 99.60±1.19along with mean % RSD 1.19 was obtained for interday accuracy and precision. Specificity was carried out by spotting blank matrix which resulted in clear Densitogram without any peak. The results observed in specificity study shows that the components of biological matrix did not interfere with the analyte, thereby confirming the specificity of the analytical method. The sensitivity of the method was determined by calculating LOD and LOQ for TER which was found to be 11.5ng / band and 34.9ng / band respectively.

Table No.3- Results of intra-day and inter-day precision and accuracy

Concentration (ng/band)	Intra-day precision				Inter-day precision
Concentration (ng/band)	Area (µV. Sec)	% Recovery ± SD	*Mean % Recovery ± SD**	% RSD*	Area (µV. Sec)	% Recovery ± SD	*Mean % Recovery ± SD**	% RSD*
100	2287	99.37 ± 0.66	99.10 ± 0.51	0.51	2310.7	100.68 ± 0.95	99.60 ± 1.19	1.19
100	2273				2292
100	2268.4				2283.4
150	2986.1	98.52 ± 0.41			2999.1	99.80 ± 0.67
150	2994.5				3028.5
150	2971.6				3014.6
200	3726	99.42 ± 0.28			3710	98.33 ± 0.31
200	3734.8				3704.8
200	3742.3				3692.3

(ng-Nano gram, n- number of readings, SD- standard deviation, RSD- Relative standard deviation)

CONCLUSION:

Development and validation of HPTLC-MS method for the analysis of Terconazole in the biological matrix have been reported. The method was accurate, reproducible, specific and also applicable for the routine analysis of pharmaceutical dosage forms. Additionally the HPTLC-MS is less time consuming, safe and economically useful for identification and quantitation of Terconazole from biological matrix.

ACKNOWLEDGEMENT:

The authors would like to thank Management of M.G.V, Principal and all the staff members of M.G.V’s Pharmacy College, Panchavati, Nasik, for their valuable contribution and sincere help in the present research work.

DECLARATION OF INTEREST:

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

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Received on 27.07.2014 Accepted on 26.08.2014

Asian J. Pharm. Ana. 4(3): July-Sept. 2014; Page 108-112