INTRODUCTION:

Benfotiamine: Benzenecarbothioic acid S-[2-[[(4-amino-2-methyl-5-pyrimidinyl)methyl]formylamino]-1-[2-(phosphenoxy)ethyl]-1-propenyl]ester;thiobenzoic acid S-ester with N-[4-amino-2-methyl-5-pyrimidinyl)methyl1]-N-(4-hydroxy-2-mercapto-1-methyl-1-butenyl)formamide O-phosphate; S-benzothialmine monophosphate is a synthetic S-acyl derivative of thiamine (vitamin B1) belonging to the family of compounds known as Allithiamines⁸. It is a lipid-soluble form of the Vitamin B1. It relieves pain from neuropathy, retinopathy, nephropathy. By blocking AGEs (Advanced Glycation End Products), it prevents some complications due to diabetes, such as blood vessel damage and atherosclerosis.

Literature survey reveals that few HPLC methods have been reported for the estimation of benfotiamine individually and combination with other drugs. Till now, there is no simple UV-visible spectroscopic method has been reported using simple and ecofriendly solvents. The main objective of the present study is to develop simple, sensitive, accurate and precise UV-visible spectroscopic method for estimation of benfotiamine in bulk and tablet dosage form. The validation has been carried out as per ICH guidelines.

Figure 1 Structure of benfotiamine

MATERIALS AND METHODS:

Materials

Benfotiamine bulk drug was obtained from Meyer Organics Pvt. Ltd. (Thane, India). Water used for the preparation of different dilutions was generated by triple stage distillation unit at parent institute. Whatman filter paper number 01 from GE Healthcare UK Ltd used for filtration purpose. The commercially available Benalgis^® tablets (B.No.SH13009 of Swiss Garnier Life Sciences Ltd., H.P) containing 100 mg of BEN were used and it was procured from the local market. The glassware’s used were class-A grade.

Instruments

Absorption spectral measurements were carried out using Shimadzu UV-1800 model UV-Visible spectrophotometer with 1cm quartz matched cuvettes. A UV probe personal spectroscopy software version 2.33 was used for instrument control and Microsoft excel 2007 for data analysis.

Preparation of standard stock solution

100 mg of BEN working standard was accurately weighed and transferred into a 100 mL volumetric flask and dissolved with minimum quantity of water. After that 0.8 ml of conc. HCl was added to dissolve the drug completely and final volume was made up to 100 mL with water. The solution was observed to contain 1000μg/mL (Stock Solution-I) of BEN. From this stock 10 mL was transferred to 100 mL volumetric flask and volume was made up to 100 mL by water to obtain 100 μg/mL (Stock Solution-II).

Selection of λ max

The solution (stock solution –II) was scanned between 400 and 200 nm using water as blank. The UV spectrum of BEN in water had shown λmax at 245 nm. Commercial tablet preparation of batch number (SH13009) was assayed which was labeled to contain 100 mg of BEN per tablet. Twenty tablets of formulation (Benalgis® tablet from Swiss Garnier Life Sciences Ltd.) containing 100 mg of BEN were accurately weighed and powdered. The powder equivalent to 100 mg of BEN was weighed and transferred to a 100 mL volumetric flask; 50 mL water was added and sonicated for 10 min. The volume was made to 100 mL with water. The solution was filtered through Whatman filter paper No. 01. From this filtrate, 10 mL was transferred to a 100 mL volumetric flask and diluted with water to 100 mL in order to obtain the concentration of 100 μg/mL. From this stock 2mL was transferred to 10 mL of water to obtain the concentration of 20 μg/mL. The absorbance was measured at 245 nm using water as blank. This procedure was repeated for six times. The amount of BEN present in formulation was calculated by comparing it with standard absorbance. The results obtained are shown in Table 1.

Method validation

The developed method was validated as per ICH guidelines (ICH Q1B, 1996, ICH Q2 R1, 1996) for following parameters

Linearity

The linearity was studied in the concentration range of 4 – 24 μg/mL at 245 nm. Linear regression data are shown in Table 2 and Figure 2 and 3.

Table 2: Optical Characteristics

Sr. No.	Parameters	Method wavelength (nm)
1	Absorption maxima (nm)	245
2	Beer’s law limit (μg/ml)	4 - 24
3	Correlation coefficient	0.9990
4	Regression equation ( y =mx + c )	y = 0.039x + 0.002
5	Slope (m)	0.039
6	Intercept (c)	0.002

Figure 2 Linearity plot of BEN in bulk at 245 nm

Figure 3 Linearity plot of BEN in dosage form at 245nm

Specificity and selectivity

The spectra obtained from tablet solutions were identical with that obtained from standard solution containing an equivalent concentration of BEN (Figure 4). This showed that there was no any interference from excipients. Therefore, it could be said that developed method is highly selective.

Figure 4 Overlay UV Spectrum of BEN in Standard and Tablet formulation.

Recovery studies

To ensure accuracy of the method, recovery studies were performed by standard addition method of known amount of samples to preanalyzed samples and subsequent solutions were reanalyzed. At each level, three determinations were performed. The absorbances were measured at 245 nm using water as blank and the amount of drug recovered from the formulation were calculated, and the results obtained are shown in Table 3.

Table 3: Results of Recovery Studies

Drug	Initial amount [µg/mL]	Amount added [µg/mL]	Amount recovered [µg/mL] [n = 3]	% Recovered
BEN	8.0	4.0	11.69	98.71
	8.0	8.0	16.15	100.93
	8.0	12.0	19.76	98.80
		Mean		99.48
		SD		1.25
		% RSD		1.26

Precision

The precision of an analytical procedure expresses the closeness of agreement (degree of scatter) between a series of measurements obtained from multiple sampling of the same homogeneous sample under the prescribed conditions. Precision of the method was determined in terms of repeatability and Intraday - Interday precisions.

Repeatability

Repeatability of the method was determined by analyzing six samples of same concentrations of drug. Absorbances were recorded. The results of this determination are reported in Table 4.

Table 4 Results of Repeatability Studies

Drugs

Amount taken

[µg/mL] [n=6]

Amount found

[µg/mL]

RSD

BEN

20.0

19.82

0.65

Intraday and Interday precision

Intraday precision was determined by analyzing the drugs at three different concentrations and each concentration for three times, on the same day. Interday precision was determined similarly, but the analysis being carried out daily, for three consecutive days. The results are summarized in Table 5

Table 5 Result of intraday and Interday precision

Drug	Conc. [µg/mL]	Intra day Amount found		Inter day Amount found
Drug	Conc. [µg/mL]	Mean ± SD	% RSD*	Mean ± SD	% RSD [n=3]
BEN	4	4.17±0.004	2.72	4.05 ± 0.003	2.25
	12	11.80±0.005	1.18	11.82 ± 0.003	0.77
	24	24.33±0.003	0.32	24.33 ± 0.004	0.51

* = Average of 3 determinations

Robustness

The robustness of a method is its capacity to remain unaffected by small but deliberate changes in conditions. To determine the robustness of the method, the experimental conditions were deliberately altered and assay was evaluated. The effect of wavelength was carried out at two different wavelength ±2 nm to actual wavelength. For changes of conditions, the sample was assayed in triplicate. When the effect of altering one set of conditions was tested, the other conditions were held constant at the optimum values. Assay of BEN for all deliberate changes of conditions was within 98–102 %. The results are shown in Table 6.

Table 6 Results from robustness study (± 2 nm of the optimum wavelength)

Method Wavelength

(nm)

Condition

(nm)

% Assay*

% RSD

245

243

247

98.20

98.33

0.19

0.34

* = Average of 3 determinations

Ruggedness

To determine ruggedness, two different analysts has performed assay on marketed tablets of the drug in similar operational and environmental conditions using developed method. The results are summarized in Table 7.

Table 7 Results of ruggedness studies

Parameters	Analyst I	Analyst II
Wavelength (nm)	245	245
% Assay*	99.23	98.84
% RSD*	0.81	0.58

* = Average of 3 determinations

Solution stability

The stability of the standard solution was tested at intervals of 1, 4 and 10 hrs. The stability of solutions was determined by comparing absorbance of BEN. The absorbance values were within 0.5% after 10 h. These results indicate the solution was stable for 10 h at ambient temperature, because there was no change in assay value. The %RSD of assay was 0.19% at 245 nm, after 10h. The results are shown in Table 8.

Table 8 Stability Data

Method Wavelength (nm)	Time (h)	% Assay*	% RSD
	1	99.35	0.34
245	4	99.23	0.19
	10	98.58	0.19

* = Average of 3 determinations

RESULTS AND DISCUSSION:

The overlay UV spectra of standard and tablet solutions of BEN in water was found to be same (Figure 2). The UV spectrum of BEN in Water has maximum absorption (λmax), at 245 nm. The absorbance of excipients in tablet solution did not interfere with BEN at 245nm. As a result, this wavelength was selected for quantitative analysis and validation. The drug obeyed Beer– Lambert’s law in the concentration range of 4–24 μg/mL with regression coefficient 0.9990 at 245nm. The overall % recovery was found to be 99.48% at 245 nm, which reflect that the method is free from interference of the impurities and other additives used in tablet formulation. Relative standard deviations of absorbance from six measurements were always less than 2%. The developed method was found to be precise as the %RSD values for Intraday and Interday precision were found to be less than 2%. The method was also found to be accurate as % recoveries were ranging from 98.71 to 100.93%.

CONCLUSION:

The developed UV spectroscopic method for the determination of Benfotiamine has the advantage of being fast, simple, inexpensive and applicable over a wide concentration range with high precision and accuracy. The developed UV spectroscopic method is cheaper, simpler and faster as compared to other electrophoretic, photometric and chromatographic methods for analysis of BEN in the pharmaceutical preparations. The method was validated as per ICH guidelines. The results of the validation tests were found to be satisfactory and therefore this method can be applied successfully to analyze drug formulations.

ACKNOWLEDGMENT:

We would like to thank Meyer organics Pvt. Ltd, Thane, India, for providing pure drug samples for this study, The Principal, Dr.(Mrs.) Supriya Shidhaye for providing us the research facility.

REFERENCES:

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Received on 14.12.2013 Accepted on 03.05.2016

Asian J. Pharm. Ana. 2016; 6(3): 133-137.

DOI: 10.5958/2231-5675.2016.00020.X