Extractive visible spectrophotometric determination of voriconazole in tablet dosage form using ion-association methods

K. Raghubabu¹, V. Jagannadharao², B. Kalyana Ramu³

¹Department of Engineering Chemistry, AU College of Engineering (A), Andhra University, Visakhapatnam -530003 Andhra Pradesh (India)

²Department of Chemistry, Anil Neerukonda Institute of Technology and Sciences, Sangivalasa (AP) India.

³Department of Chemistry, Maharajah’s College (Aided and Autonomous), Vizianagaram-535002 (AP) India

*Corresponding Author E-mail: drraghualways@yahoo.co.in; kalyanaramu23566@gmail.com

ABSTRACT:

Two simple and sensitive extractive visible spectrophotometric methods (A and B) for the assay of voriconazole in pure and dosage forms based on the formation of colored chloroform soluble ion-association associates under specified experimental conditions are described. Two dyes namely acidic dye Tropaeoline ooo (TPooo, method A), Azo carmine- G (ACG, method B) are utilized. The extracts of the ion-associates exhibit absorption maxima at 500nm and 550nm for methods A and B respectively. Regression analysis of Beer-Lambert plots showed good correlation in the concentration ranges (5.0-25) µg/ml for method A, (10-50) µg/ml for method B respectively. The proposed methods are applied to commercial available tablets and the results are statistically compared with those obtained by the UV reference method and validated by recovery studies. The results are found satisfactory and reproducible. These methods are applied successfully for the estimation of the voriconazole in the presence of other ingredients that are usually present in dosage forms. These methods offer the advantages of rapidity, simplicity and sensitivity and low cost and can be easily applied to resource-poor settings without the need for expensive instrumentation and reagents.

KEYWORDS: Antifungal, ACG, Assay, Ion-Association methods, Statistical analysis, TPOOO

1INTRODUCTION:

Voriconazole (VCZ) (2R, 3S)-(2, 4-difluorophenyl)-3-(5-fluoro-4-pyrimidinyl)-1-(1H-1, 2, 4-triazole-1-yl)-2-butanol [Figure1] is a newest second generation drug that belongs to triazole antifungal agent ^[1-2] used in the treatment of armamentarium fungal infections. This medication is indicated for the primary treatment of acute invasive aspergillosis and salvage therapy for rare but serious fungal infections caused by Scedosporium apiospermum and Fusarium Spp. The drug is a with structure related to that of fluconazole and a spectrum of activity comparable to that of itraconazole acting by inhibiting fungal ergosterol biosynthesis. This inhibition is more selective for fungal than for mammalian enzyme systems. Its primary mode of action is by inhibition of the fungal cytochrome P-450-dependent 14 alpha-sterol demethylase, an essential enzyme in fungal ergosterol biosynthesis. The accumulation of 14-alpha methyl sterols correlates with the subsequent loss of ergosterol in the fungal cell wall and may be responsible for the antifungal activity of voriconazole.

The use of this drug is increasing, because it represents an alternative to amphotericin B treatment in invasive fungal infections. The drug is not yet official in any pharmacopoeia.

Figure 1: Chemical structure of Voriconazole

Several analytical techniques like HPLC^[3-15], HPLC and first order derivative spectrophotometric method ^[16], LC methods ^[17-18], capillary electrophoresis and LC method ^[19], HPTLC^[20-21], LC-MS-MS^[22-23], LC-EI-MS^[24], LC-APCI-MS/MS^[25] and UV spectrophotometry ^[26],

Differential pulse Polarography (DPP)^[27] have been reported in the literature. But to the best of our knowledge there is no single method available for the estimation by visible spectrophotometry which is far simpler and economical and less time consuming as compared to above mentioned methods. So the authors have made some attempts in developing visible spectrophotometric methods and succeeded in developing two methods based on the reaction between the drug and acidic dyes namely TPooo or ACG under specified experimental conditions.

As the extraction spectrophotometric procedures are popular for their sensitivity and selectivity in the assay of drugs, the extractive spectrophotometric acid- dye technique ²⁸ was therefore, utilized in the present work for the estimation of VCZ. The present paper describes two simple and sensitive extraction visible spectrophotometric methods for the determination of VCZ, based on its tendency to form chloroform extractable ion-associates with acidic dye TP ooo (C.I No.14600) belonging to azo category dye (method A) or Azocarmine G (C.I No.50085) belonging to Phenazine category dye (method B) under experimental conditions by exploiting the basic nature (nitrogen in triazole linked to 1-butan-2-ol) of the drug molecule. According to the literature, it is the first time for VCZ determination in formulations by visible spectrophotometry.

The proposed methods for VCZ determination have many advantages over other analytical methods due to its rapidity, lower cost and environmental safety. Unlike HPLC, HPTLC procedures, the instrument is simple and is not costly. Economically, all the analytical reagents are inexpensive and available in any analytical laboratory. The proposed methods report a new for the determination of VCZ in pharmaceuticals. These methods can be extended for the routine assay of VCZ formulations.

MATERIALS AND METHODS:

Apparatus and chemicals

A Shimadzu UV-Visible spectrophotometer 1601 with1cm matched quartz cells was used for all spectral measurements. A Systronics digital pH meter mode-361 was used for pH measurements. All the chemicals used were of analytical grade. Pure VCZ drug was obtained as a gift sample from Dr Reddy laboratories, Hyderabad, AP (India). Vfend-50mg tablets and Vonaz-50mg tablets were purchased from local market. Tropaeolin 000 (Fluka, 0.2%, 5.7x10^-3M prepared by dissolving 200mg of Tropaeolin 000 in 100ml distilled water and subsequently washed with chloroform to remove chloroform soluble impurities), 0.1M HCl (prepared by diluting 8.7ml of Con. Hydrochloric acid to 1000ml with distilled water and standardized) ACG solution ( Gurr, 0.05%, 8.75x10^-4M prepared by dissolving 50mg of azocarmine G in 100ml of distilled water containing traces of sodium hydroxide and subsequently washed with chloroform to remove chloroform soluble impurities), pH 1.5 Buffer solution (prepared by mixing 289ml of 0.1M glycine solution(7.507g of glycine and 5.85 g NaCl was dissolved in 100ml of distilled water) with 711 ml of 0.1M HCl and the pH of the solution was adjusted to 1.5) were prepared.

Preparation of Standard stock solution: The standard stock solution (1mg/ml) of VCZ was prepared by dissolving 100mg of VCZ in 100 ml distilled water. The working standard solutions of VCZ were obtained by appropriately diluting the standard stock solution with the same solvent.

Preparation of Sample solution: About 10 tablets were pulverized and the powder equivalent to 100mg of VCZ was weighed, dispersed in 25ml of IPA, sonicated for 30 minutes and filtered through Whatman filter paper No 41.The filtrate was evaporated to dryness and the residue was dissolved as under standard solution preparation.

Fig.2: Absorption spectra of VCZ-TPOOO

Fig.3: Absorption spectra of VCZ-ACG

Fig.4: Beer’s law plot of VCZ-Tpooo

Fig.5: Beer’s law plot of VCZ-ACG

Assay: Aliquots of the standard VCZ solution [1.0-5.0 ml, 50µg/ml (method A) and 1.0-5.0ml, 100µg/ml (method B)] were placed in a series 125ml separating funnels. Then 6.0ml of 0.1M HCl and 2.0ml of TPooo solution 5.70x10^-3M(for method A) or 6.0 ml of pH 1.5 buffer solution and 2.0 ml of ACG solution (8.75x10^-4M)(for method B) were added. The total volume of aqueous phase in each separating funnel was adjusted to 15.0ml with distilled water. Then 10.0ml of chloroform was added to each separating funnel and the contents were shaken for 2 minutes. The two phases were allowed to separate. The absorbance of the separated chloroform layer were measured at 500nm (method A) or 550 nm (method B) (Fig. 2and3 showing absorption spectra) against a reagent blank within the stability period (5 minutes to 1hour). The amount of drug was computed from its calibration graph (Fig.4and5).

Fig. 7: Probable scheme of reaction for method B

RESULTS AND DISCUSSION:

Optimum operating conditions used in the procedure were established by adopting variation of one variable at a time (OVAT) method. The effect of various parameters such as time, volume and strength of TPooo, ACG reagents, 0.1M HCl, pH buffer solutions and solvent for final dilution of the colored species were studied. TPooo and ACG were preferred for this investigation as they yield high molar absorptivity values among six dyes belonging to different chemical classes. The water immiscible solvents tested for the extraction of colored complex into organic phase include chloro benzene, dichloromethane, carbon tetrachloride, benzene, nitro benzene, n-butanol or chloroform. Chloroform was preferred for its selective extraction of colored drug -dye complex into organic layer from the aqueous phase. The stoichiometric ratio of the dye-drug was determined by the slope ratio method and was found to be 1:1 for methods A and B respectively. The optical characteristics such as Beer’s law limit, Sandell‘s sensitivity, molar absorptivity, percent relative standard deviation, (calculated from the six measurements containing 3/4^th of the amount of the upper Beer’s law limits), Regression characteristics like standard deviation of slope (S_b), standard deviation of intercept (S_a), standard error of estimation (S_e) and % range of error (0.05 and 0.01 confidence limits) were calculated and the results are summarized in Table-1. Commercial formulations containing VCZ were successfully analyzed by the proposed methods. The values obtained by the proposed and reference methods for formulations were compared statistically by the t-and F-test and found not to differ significantly. As an additional demonstration of accuracy, recovery experiments were performed by adding a fixed amount of the drug to the pre analyzed formulations at three different concentration levels. These results are summarized in Table-2.

Table 1: Optical characteristics, precision and accuracy of proposed methods

Parameters	Method A	Method B
λ max (nm)	500	550
Beer’s law limit (µg/ml)	5- 25	10-50
Sandell’s sensitivity (µg/cm²/0.001 abs. unit)	8.237x10^-4	1.434x10^-3
Molar absorptivity (Litre/mole/cm)	4.24x10⁵	2.435x10⁵
Regression equation (Y) ^*= a +b c
Intercept (a)	-0.003	0.014
Slope(b)	0.124	0.066
%RSD	1.789	1.91
% Range of errors (95% Confidence limits) 0.05 significance level 0.01 significance level	1.878	2.004
	2.945	3.144

*Y= a + b c; Where Y= absorbance, c= concentration of VCZ in µg/ml.

Chemistry of colored species: The protenated nitrogen (positive charge) of the drug molecule in acid medium is expected to attract the oppositely charged part (negative charge) of the dye and behave as a single unit being held together by electrostatic attraction as given in scheme (Fig.6and7).

CONCLUSION:

A significant advantage of an extraction spectrophotometric determination is that it can be applied to the determination of individual compounds in a multi component mixture. This aspect of spectrophotometric analysis is of major interest in analytical chemistry, since, it offers distinct possibilities in assay of a particular component in a complex dosage formulation. In the present study, VCZ was determined successfully as pure compound as well as a single component in representative dosage formulations. The proposed methods applicable for the assay of drug and the advantage of wider range under Beer’s law limits. The proposed extractive visible spectrophotometric methods are validated as per ICH guide lines and possess reasonable precision, accuracy, simple, sensitive and the proposed methods report a new for the determination of VCZ in pharmaceuticals. These methods can be extended for the routine assay of VCZ formulations

Table 2: analysis of voriconazole in pharmaceutical formulations by proposed and reference methods.

Method	*Formulations	Labeled Amount (mg)	Found by Proposed Methods			Found by Reference Method ± SD	#% Recovery by Proposed Method ± SD
Method	*Formulations	Labeled Amount (mg)	**Amount found ± SD	t	F	Found by Reference Method ± SD	#% Recovery by Proposed Method ± SD
A	Batch-1	50	49.669 ± 0.063	1.23	1.59	49.728 ± 0.049	99.34 ± 0.126
A	Batch-2	50	49.551 ± 0.153	0.87	1.54	49.457± 0.153	99.10 ± 0.306
B	Batch-1	50	49.673 ± 0.089	0.71	1.53	49.728 ± 0.049	99.407 ± 0.178
B	Batch-2	50	49.622 ± 0.152	1.29	1.10	49.457± 0.153	99.244 ± 0 .304

* Different batches from two different companies (Batch-1 Vfend tablets of Pfizer, Batch 2: Vonaz tablets of United Biotech)

**Average ± Standard deviation of six determinations, the t- and F-values refer to comparison of the proposed method with reference method (UV). Theoretical values at 95% confidence limits t =2.57 and F = 5.05.

# Recovery of 10mg added to the pre-analyzed sample (average of three determinations). Reference method (reported UV method) using double distilled water (λ _max=255nm).

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Received on 20.03.2012 Modified on 30.03.2012

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