Zero order and Area under Curve Spectrophotometric Methods for Determination of Riboflavin in Pharmaceutical Formulation

Audumbar Mali¹*, Seeta Mali², Ritesh Bathe¹, Manojkumar Patil¹, Ashpak Tamboli²

¹Department of Pharmaceutics, Sahyadri College of Pharmacy, Methwade,

Sangola-413307, Solapur, Maharashtra, India.

²Department of Pharmaceutical Chemistry, Sahyadri College of Pharmacy, Methwade,

Sangola-413307, Solapur, Maharashtra, India.

*Corresponding Author E-mail: maliaudu442@gmail.com

ABSTRACT:

Simple, fast and reliable spectrophotometric methods were developed for determination of Riboflavin in bulk and pharmaceutical dosage forms. The solutions of standard and the sample were prepared in Methanol. The quantitative determination of the drug was carried out using the zero order derivative values measured at 445 nm and the area under the curve method values measured at 420-473nm (n=2). Calibration graphs constructed at their wavelengths of determination were linear in the concentration range of Riboflavin using 5-25μg/ml (r²=0.999 and r²=0.999) for zero order and area under the curve spectrophotometric method. All the proposed methods have been extensively validated as per ICH guidelines. There was no significant difference between the performance of the proposed methods regarding the mean values and standard deviations. Developed spectrophotometric methods in this study are simple, accurate, precise and sensitive to assay of Riboflavin in tablets.

KEYWORDS: Riboflavin, UV visible spectrophotometry, AUC, Method Validation, Zero order method.

INTRODUCTION:

Riboflavin or Vitamin B2 or vitamin G is chemically, 3, 10-dihydro-7, 8-dimethyl-10-[2S, 3S, 4R)-2, 3, 4, 5 tetra hydroxyl pentyl]-benzopteridine-2, 4-Dione. It is a yellow to orange-yellow crystalline compound. Action and physiological role of vitamin B2, flavinadenine dinucleotide (FAD) and flavin mononucleotide (FMN) are coenzyme for flavor proteins involved in many oxidation-reduction reactions. Characteristic lesions of vitamin B2 deficiency are angular stomatitis; sore and raw tongue, lips, throat, ulcers in mouth; vascularization of cornea. Dry scaly skin, loss of hair; anemia and neuropathy develop later. [1, 2]

Literature survey revealed several analytical methods UV spectrophotometry [3-5] and HPLC [6-10] have been reported in bulk, pharmaceutical dosage form for determination of Riboflavin. To our notice, so far no UV- spectrophotometric method using Zero Order and Area under Curve (AUC) has been reported for the determination of Riboflavin in bulk and tablets. Hence an attempt has been made to develop new Zero Order and Area under Curve Spectrophotometric method for estimation of Riboflavin in bulk and pharmaceutical formulations with good accuracy simplicity, precision and economy.

Molecular formula: C₁₇H₂₀ N₄O₆.

Molecular weight: 376.37g/mol

Fig. 1: Chemical Structure of Riboflavin

MATERIALS AND METHODS:-

Apparatus and instrumentation

A shimadzu 1800 UV/VIS double beam spectrophotometer with 1cm matched quartz cells was used for all spectral measurements. Single Pan Electronic balance (CONTECH, CA 223, India) was used for weighing purpose. Sonication of the solutions was carried out using an Ultrasonic Cleaning Bath (Spectra lab UCB 40, India).Calibrated volumetric glassware (Borosil®) was used for the validation study.

Materials:

Reference standard of Riboflavin API was supplied as gift sample by Cipla Pharmaceutical Limited, Pune. Tablets sample with label claim 100 mg per tablet were purchased from local market Pune.

Method Development: [11-15]

Preparation of Standard and Sample Solutions:-

Stock solution of 10 μg/ml of Riboflavin was prepared in Methanol, for zero order and area under the curve spectrophotometric analysis. The standard solutions were prepared by dilution of the stock solution with methanol in a concentration range of 5, 10, 15, 20, and 25 μg/ml with methanol for zero order and area under the curve spectrophotometric methods. Methanol was used as a blank solution.

Fig. 2: Zero order derivative spectrum of Riboflavin in Methanol (20µg/ml).

Fig. 3: UV AUC spectrum of Riboflavin Methanol (20µg/ml).

Area under curve (Area calculation):-

Area under curve method involves the calculation of integrated value of absorbance with respect to the wavelength between two selected wavelengths such as λ1 and λ2 representing start and end point of curve region. The area under curve between λ1 and λ2 was calculated using UV probe software. In this study area was integrated between wavelength ranges from 420 to 473 nm.

Area calculation: (α+β) =

Where, α is area of portion bounded by curve data and a straight line connecting the start and end point, β is the area of portion bounded by a straight line connecting the start and end point on curve data and horizontal axis, λ1 and λ2 are wavelength range start and end point of curve region.

Assay Procedure:-

Twenty tablets each containing 100 mg of Riboflavin were weighed crushed to powder and average weight was calculated. Powder equivalent to 10mg of Riboflavin was transferred in 100 ml of volumetric flask. A 50 ml of methanol was added and sonicated for 15minutes. Then solution was further diluted up to the mark with methanol. The solution was filtered using Whatmann filter paper no. 41; first 5 ml of filtrate was discarded. This solution was further diluted to obtain 15µg/mL solution with water subjected for UV analysis using methanol as blank. Appropriate dilutions were made with methanol from stock solution for both zero order and area under the curve spectrophotometric methods.

RESULTS AND DISCUSSION:-

The zero order and area under the curve spectra for Riboflavin were recorded at the wavelength of 445nm and 420-473 nm respectively [Fig. 2 and 3].

Linearity and Range:

Under the experimental conditions described, the graph obtained for zero order and area under the curve spectra showed linear relationship. Regression analysis was made for the slope, intercept and correlation coefficient values. The regression equations of calibration curves were Y=0.012x-0.004 (r²=0.999) at 445nm for zero order derivative spectrophotometry and Y=0.09x-0.021 (r²=0.999) at 420-473nm for area under the curve spectrophotometry. The range was found to be 5-25μg/ml for both zero order and area under the curve spectrophotometric methods.

Table 1: Assay of tablet dosage form.

Sr.No.	Sample Solution Concentration (µg/ml)	*Amount found (%) Zero derivative**	*Amount found (%) AUC**	Mean % Found zero derivative	Mean % Found AUC	%RSD zero derivative	% RSD AUC
1	15	100.35	98.23
2	15	99.84	102.39	100.92	100.60	0.6320	0.6892
3	15	102.57	101.19

*n=3, % RSD = % Relative Standard Deviation.

Fig. 4: Zero order derivative spectrum of Riboflavin dosage form (25µg/ml).

Fig. 5: Linearity of Riboflavin by Absorbance

Fig. 6: Linearity of Riboflavin by AUC.

Fig. 7: Zero order derivatives overlay of Riboflavin different Concentration.

Table 3: Accuracy results for Riboflavin

Accuracy level	Sample conc (µg/)	Std. conc	Total amnt. Added (µg/m)	%Recovery zero derivatie	% Recovery AUC*	Mean of Zero derivative*	Mean of AUC	% RSD Zero derivative	% RSD AUC
80	15	12	27	98.15	98.66
100	15	15	30	102.54	100.11	100.32	99.98	0.5423	0.5981
120	15	18	33	100.29	101.18

*n=3, % RSD = % Relative Standard Deviation.

Table 4: Results of Intra and Inter Day Precision

Parameters	Intra Day Precision		Inter Day Precision
Parameters	S.D*	% RSD*	S.D*	% RSD*
Zero derivative	0.0046	0.6347	0.0036	0.6120
Area under the curve	0.7439	0.5123	0.8431	0.4972

Precision:

To determine the precision of the method, Riboflavin solutions at a concentration of 10μg/ml were analysed each three times for both zero order and area under the curve spectrophotometric methods. Solutions for the standard curves were prepared fresh everyday. [16-18]

'Sensitivity:

The limit of detection (LOD) and limit of quantification (LOQ) were calculated by using the equations LOD = 3xσ/ S and LOQ = 10xσ/S, where σ is the standard deviation of intercept, S is the slope. The LOD and LOQ were found to be 0.4831μg/ml and 1.4482μg/ml respectively for zero order derivative and The LOD and LOQ were found to be 0.5201µg/ml &1.5608µg/ml for area under the curve methods respectively. [19-21]

Analysis of the Marketed Formulation:

There was no interference from the excipients commonly present in the tablets. The drug content was found to be 100.92% and 100.60% zero order and area under the curve spectrophotometric methods respectively. It may therefore be inferred that degradation of Riboflavin had not occurred in the marketed formulations that were analysed by this method. The low % R.S.D. value indicated the suitability of this method for routine analysis of Riboflavin in pharmaceutical dosage form. [19-21]

Table 5: Summary of validation parameters

Parameter	Zero derivative	AUC
λ range	200-400 nm	200-400 nm
Regression Equation (Y=mx+c)	Y=0.012x-0.004	Y=0.09x-0.021
Measured wavelength	445nm	420-473nm
Linearity range	5-25µg/ml	5-25µg/ml
Slope	0.012	0.09
Intercept	0.004	0.021
Correlation coefficient (R²)	0.999	0.999
Limit of Detection (LOD) µg/ml	0.4831	0.5201
Limit of Quantitation (LOQ) µg/ml	1.4482	1.5608
Accuracy (Mean % Recovery)	100.32	99.98
Precision (% RSD)	0.5423	0.5981

CONCLUSION:

No UV or Area under Curve spectrophotometric methods have been described for the determination of Riboflavin. Therefore simple, fast and reliable derivative spectrophotometric methods were developed for the routine determination of Riboflavin. The developed methods can be concluded as accurate, sensitive and precise and can be easily applied to the pharmaceutical formulation.

ACKNOWLEDGEMENT:

The authors are highly thankful to the Sahyadri College of Pharmacy, Methwade, Sangola, Solapur, Maharashtra, India for proving all the facilities to carry out the research work.

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Received on 16.01.2016 Accepted on 02.02.2016

Asian J. Pharm. Ana. 6(1): January- March, 2016; Page 35-40

DOI: 10.5958/2231-5675.2016.00006.5

Parameters	Zero order derivative	Area Under the Curve
Linearity range (µg/ml)*	5-25	5-25
r²± S.D*	0.999	0.999