Estimation of Voriconazole in Biological Matrix by HPTLC-MS

Santosh R. Tambe¹*, Sanjay D. Sawant²

¹Assistant Professor, M. G. V’s Pharmacy College, Panchavati, Nashik

²Professor and Principal, STES SKN College of Pharmacy, Kondhwa (BK), Pune – 411 048

*Corresponding Author E-mail: santoshsrt.mgv@gmail.com

ABSTRACT:

A High Performance Thin Layer Chromatography Tandem Mass Spectrometry (HPTLC-MS) method was developed for estimation of Voriconazole in a biological matrix. HPTLC-MS is a rapid and economic technique for appropriate identification and quantitation of active pharmaceutical ingredients in pharmaceutical dosage forms and also biological matrices. The Rf value of the drug was 0.33 ± 0.011 using Toluene: Ethyl Acetate: Methanol: (6:0.5:2.5 v/v/v) as the mobile phase. Linearity was obtained within a concentration range of 100 – 600 ng/band with regression coefficient 0.999. The accuracy of the proposed method for analyzing the API in terms of percentage recovery was 100.1 with percentage RSD 1.53%, both of which were within the specified limits, verifying that the method is accurate and precise. LOD and LOQ were 20. 47 ng/band and 62.05 ng/band, respectively. The recovery after application to biological matrix was 97.7± 1.46 % proving its applicability to biological analysis. Specificity study showed that the components of biological matrix did not interfere with the analyte, thereby confirming the specificity of the method. Mass spectrometric analysis of the samples resulted in a spectrum showing molecular ion peak at 350.1 m/z. HPTLC-MS method resulted in direct confirmation in identifying the drug. . This HPTLC method seems to be convenient as well as less time consuming and thus illustrating its wide applicability.

KEY WORDS: HPTLC-MS, Planer Chromatography, Mass spectrometry, Voriconazole.

INTRODUCTION:

Voriconazole (VOR) (Fig. 1), a derivative of Fluconazole, is a second-generation triazole antifungal agent. VOR is chemically (2R, 3S)-2-(2, 4-difluorophenyl)-3-(5-fluoro-4-pyrimidinyl)-1-(1H-1, 2, 4-triazol-1-yl)-2-butanol. The primary mode of action of VOR is the inhibition of fungal cytochrome P-450-mediated 14-α-lanosterol demethylation, an essential step in fungal ergosterol biosynthesis. The accumulation of 14 alpha-methyl sterols correlates with the subsequent loss of ergosterol in the fungal cell wall and this may be responsible for the antifungal activity of VOR¹. It has been shown to be more selective for fungal cytochrome P-450 enzymes than for various mammalian cytochrome P-450 enzyme systems^{1, 2}.

VOR is used to treat serious fungal infections such as invasive aspergillosis (a fungal infection that begins in the lungs and spreads through the bloodstream to other organs) and esophageal candidiasis (infection by a yeast-like fungus that may cause white patching in the mouth and throat). It works by slowing the growth of the fungi that causes infection^{3, 4}.

Figure 1- Structure of VOR

Literature survey revealed number of methods available for estimation of VOR which includes UV Spectrophotometric methods^5-7, High Performance Liquid Chromatographic methods^8-14, Stability-indicating HPLC method¹⁵, Experimental design approach in HPLC method¹⁶, along with some modification in the methods such as RP-HPLC^17,18. Also some advanced methods like LC-MS/MS^19-21, UPLC method²², Stability indicating UPLC method²³ and Ultra-Performance Liquid Chromatography–Tandem Mass Spectrometry Method²⁴ are reported.

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-to-operate, planar chromatographic technique that has been used routinely in general chemistry laboratories since several decades to separate chemical and biochemical compounds. Traditionally, chemical and optical methods are employed to visualize the analyte spots on the TLC plate However, direct identification and structural characterization of the analytes on the TLC plate are not possible through these methods. Hence, there has been long held interest in the development of interfaces that allow TLC to be combined with mass spectrometry (MS); one of the most efficient analytical tool for structural elucidation²⁵. This method is very rapid as well as economical 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 CATS software, and Hamilton syringe (100 µL), 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, Chloroform and Toluene (AR grade, Fisher Scientific, India) were used for mobile phase preparation.

Preparation of standard and sample solution

Accurately weighed 10 mg of drug was transferred to 10 mL volumetric flask and diluted to 10 mL using methanol to get concentration 1 mg/mL. From this stock solution, 1 mL was further diluted to 10 mL using methanol.

Preparation of matrix for analysis

For the analysis of the drug from a biological matrix, a simulated fluid was prepared in the laboratory; the compositions of which is given below in Table 1

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 (6:0.5: 2.5v/v/v). TLC plates were prewashed with methanol and activated in an oven at 110 ºC 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 vapors of mobile phase for 15 min. The developed TLC plate was air dried and then scanned from 200 to 400 nm using Camag TLC scanner 3 using WinCATS software. The drug showed reasonably good response at 253 nm keeping the slit dimension of 6.00 x 0.30 mm and scanning speed of 20 mm/s.

Table 1- Master formula for biological matrix

Ingredients	Quantity (g)
Glucose	2.2065
Lactic Acid	0.5
Acetic Acid	0.013
Albumin	0.2164
Urea	1.0432
Sodium chloride	0.2621
Potassium chloride	0.2485
Calcium Chloride	0.026
Glycerol	0.1
Distilled water	up to 250 mL

Linearity and calibration graph

Linearity was obtained over the concentration range 100 – 600 ng/band by applying 1 – 6 µL from standard solution (100ng/µL) as a band of 6 mm width on the TLC plate. All the chromatographic conditions were maintained as above. The R_f value for VOR was 0.33 ± 0.011 along with asymmetry factor 1.2 (Fig 2). Peak areas of VOR were plotted against corresponding concentrations and least square regression analysis was performed to generate the calibration equation (Fig 3).

Figure 3- Calibration graph of VOR

Application of the proposed method for estimation of VOR in biological matrix

Appropriate volume (4 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 400 µg/mL was used for estimation of VOR in biological matrix.

Spiked samples (100 ng/μL, 6 replicate) were prepared by adding 0.25 mL solution from above standard solution of VOR (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 2.

Mass Spectrometric 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 standard of polypropylene glycol which was supplied by AB SCIEX using a 1mL Hamilton syringe and monitored 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 470 °C. The drug detection and mass identification were performed. Q1 and Q3 quadrupoles were set to unit resolution. The mass spectrum of VOR was displayed as reported in Fig. 4.

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 VOR in biological matrix. The run consisted of a three replicates of each 200, 300, 400 ng/band of VOR. The overall precision of the method expressed as relative standard deviation and recovery of the method. Intraday accuracy ranged from 101.69 % to 98.61 % and precision was 1.57 %. Inter day accuracy ranged from 101.92 % to 99.12 % and precision was 1.50 %. The mean Recovery, standard deviation (SD), coefficient of variation (% RSD) was evaluated and their results were tabulated in Table 3.

Figure 4- Full scan mass spectrum of VOR

Table 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*
200	2741.5	101.69 ± 0.84	99.99 ± 1.57	1.57	2743.5	101.92 ± 0.89	100.21 ± 1.50	1.50
200	2724.9				2728.9
200	2754.2				2760.2
300	3590.2	99.66 ± 0.59			3579.2	99.58 ± 0.65
300	3560.1				3557.1
300	3582.7				3590.7
400	4396.5	98.61 ± 0.69			4399.5	99.12 ± 0.92
400	4404.1				4464.1
400	4441.7				4431.7

Figure 5- Densitogram of Blank sample of matrix

· 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. 5 where, a blank sample of matrix was spotted on the plate. The densitogram shows that there were no endogenous peaks at the retention time of VOR. The components of biological matrix did not interfere with the analyte, thereby confirming the specificity of the analytical method.

· LOD and LOQ:

The limit of detection and limit of Quantitation were calculated for VOR by using the standard deviation and slope obtained from linearity studies and it was found to be LOD 20.47 ng / band and LOQ 62.05 ng / band.

RESULTS AND DISCUSSION:

The reported method is based on the principle of chromatographic separation and spectrometric identification of VOR in the biological matrix. VOR showed R_f value 0.33 ± 0.011 along with asymmetry factor 1.2 using Toluene: Ethyl Acetate: Methanol (6:0.5:2.5 v/v/v) as mobile phase. Linearity was obtained within concentration range 100 – 600 ng/band with regression coefficient 0.999. Sample recovery was found to be 97.7 ± 1.4 % when the method was applied to biological matrix. Mass spectroscopy was resulted in the spectra showing molecular ion peak at 350.1 m/z indicating mass of VOR.

The developed HPTLC 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 99.99 ± 1.57 % with mean percentage RSD 1.57 % for intraday accuracy and precision. Similarly, mean percentage recovery 100.21 ± 1.50 % along with mean percentage RSD 1.50 % was obtained for interday accuracy and precision. Specificity was carried out by spotting blank matrix which resulted in clear densitogram without any peak in the region where VOR showed a peak. The results observed in specificity study showed 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 VOR which was found to be 20.47 ng/band and 62.05 ng/band, respectively.

CONCLUSION:

Development and validation of HPTLC method for the analysis of Voriconazole in the biological matrix have been reported. The method was accurate, reproducible, specific and applicable for biological analysis. Additionally, the HPTLC-MS, a less time consuming method, for identification the drug was also developed to authenticate the analyte.

ACKNOWLEDGEMENT:

The authors would like to thank Management, Principal and all the staff members of M.G.V’s Pharmacy College, Panchavati, Nashik, and Dr. Santosh Gandhi, S. S. P. M. S. College of Pharmacy, Pune, for their valuable support and sincere help in the present research work.

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Received on 07.12.2013 Modified on 15.12.2013

Asian J. Research Chem. 7(2): February 2014; Page 166-170

Concentration (ng/band)	Area (µV. Sec) (Average, n = 6)	*Mean % Recovery ± SD**	% RSD*
200	2670.70	97.7 ± 1.46	1.49