UV Spectrophotometric Quantification of Niacinamide in Pharmaceutical Dosage Form by Multivariate Calibration Technique

 

Subhash Varun Kumar V, Kavitha J*, Lakshmi KS

Department of Pharmaceutical Analysis, SRM College of Pharmacy, SRM Institute of Science and Technology, Kattankulathur-603203, Kancheepuram District, Tamil Nadu, India.

 

ABSTRACT:

The quantification of Niacinamide in pharmaceutical dosage form employing Multivariate Calibration Technique is detailed in the current study. The λmax of Niacinamide was found to be 262 nm. The linearity of the developed method was accessed at five different concentrations ranging from of 8-24µg/mL. Correlation coefficient (r2) value was found to be above 0.998.  The repeatability of the method was predicted by performing Intraday and Interday precision studies. The developed method was validated according to ICH guidelines and results proved that the developed method was simple, linear, precise, accurate and sensitive. The developed and validated Multivariate Calibration Technique can be further employed and released into QC for the quantification of Niacinamide in pharmaceutical dosage form.

 

 

 

INTRODUCTION:

Niacinamide (Figure 1) is a vitamin B3 nutrient. It has ability to treat aging skin¹ and it is a chemically pyridine-3-carboxylic acid which appears to be a colorless solid and is water soluble². It is converted to co-factors such as NADH and NADPH and is involved in many biochemical reactions³. It is an anti-acne drug having an anti-inflammatory effect which acts by reducing the inflammation⁴. Niacinamide is the precursor of Niacinamide adenine dinucleotide (NAD) and its phosphorylated derivative (NADP)⁵. In liver, Nicotinic acid is converted to Niacinamide during the metabolism⁶. Niacinamide is sourced from the diet and a lack of this vitamin results in a diseased condition called pellagra⁷. Niacin and Niacinamide are two important supplements reported in the human food industry and also in maintaining cholesterol levels⁸. Niacinamide do not interfere above 300nm⁹. Due to its above listed important the analysis of Niacinamide plays a major role. It is official in Indian¹⁰, United States¹¹, European¹² and British¹³ Pharmacopoeias.

 

On survey, various literatures pertaining to the analysis of Niacinamide in various pharmaceutical formulation, food and biological samples employing different analytical techniques such as RP-HPLC^14,15, HPTLC¹⁶, LC-MS/MS¹⁷, UV- Spectroscopy¹⁸, Flow injection TLC¹⁹, Capillary electrphoresis²⁰, tandem Mass Spectrometry²¹ have already been reported. The present work was framed with the target to develop simple, accurate, precise and sensitive with rapid analytical method for the quantification of Niacinamide amidst the already reported complex analytical methods. Based on the above statement, a simple analytical method employing UV spectrophotometry aided multivariate calibration technique was proposed to be developed^22-25.

 

Figure 1: Niacinamide Chemical Structure

 

Multivariate calibration technique was used to predict the unknown value of Y from the available X value. Calibration is described as a mathematical function between the measured variable X and a dependent variable Y²⁶. The technique employs the use of several variables simultaneously to quantify one or many target variables. The method is inexpensive and can be applied for the routine quantification of bulk drug and pharmaceutical formulations. This technique uses simple mathematical tool for the examination of drug in the pharmaceutical dosage form²⁷.

 

If the absorbance of an analyte (X) is consistent at five selected wavelengths (λ= 266, 264, 262, 260 and 258 nm), the following equation can be transcribed for the individual selected wavelength²⁸.

 

Multivariate Calibration Technique performs the conversion of single common species analysis from one dependent variable to “m” dependent variables²⁹.

1.     Aλ₂₆₆= aXCx+k1                                                            (1)

2.     Aλ₂₆₄= bXCx+k2                                                            (2)

3.     Aλ₂₆₂= cXCx+k3                                                            (3)

4.     Aλ₂₆₀= dXCx+k4                                                            (4)

5.     Aλ₂₅₈= eXCx+k5                                                            (5)

 

Where, Aλ is the absorbance of the analyte; a, b, c, d, and e are the slopes of the linear regression line; k1, k2,k3, k4 and k5are the intercept values and C_x represents the concentration of the unknown analyte. The equation systems (1-5) can be summarized as follows,

AT= Cx(a+b+c+d+e)+KT                                                 (6)

 

Where, AT and KT represents the sum of the absorbance and intercepts of regression equations at five wavelength set, respectively. The concentration of the analyte X in a solution can be calculated using the equation:

Cx = AT – KT / (a + b + c + d + e)                                   (7)

 

EXPERIMENTS:

Chemicals and Solvents:

·       Distilled water

·       Niacinamide-The reference standard and the pharmaceutical dosage form (Healthvit tablet containing 500mg of Niacinamide) of Niacinamide was procured from the local markets.

 

Solubility:

·       Freely soluble in water, methanol, ethanol and ethyl acetate.

 

Instrumentation:

·       UV - Visible Spectrophotometer (Labindia UV-3092)

·       Ultra Sonic Bath (ILE, ILTC)

·       Electronic balance ( Shimadzu Type: BL-220H)

 

Method Development:

Solvent Selection:

Niacinamide was found readily soluble in water hence distilled water was employed as the solvent for solubilization throughout the process of analysis.

Preparation of Stock Solutions:

Standard stock solution:

The stock solution of Niacinamide (1mg/mL) was prepared by dissolving 10mg of the reference standard of the drug in 10mL of the selected solvent. The above stock solution was suitably diluted with the solvent for further analysis.

 

Determination of λ_max:

A standard solution containing 10µg/mL of Niacinamide was prepared by diluting the standard stock solution with distilled water and the diluted solution was scanned in the UV region of 200 – 400nm to record its UV spectra and the absorption maxima of the drug was determined which is represented in Figure 2.

 

 

Figure 2: UV spectra of Niacinamide

 

Preparation of sample solution:

A total of 20 tablets of Niacinamide procured from the local market were weighed and their average weight was determined. The tablets were further crushed, made as a fine powder and the contents were mixed uniformly. A weight equivalent to 10mg of the drug was accurately weighed and transferred into a 10Ml standard flask. To this 5mL of the solvent was added and the mixture was sonicated for 15mins. The volume of the above solution was made up to the make, mixed well, filtered and the filterate was suitably diluted for further use employing water as solvent.

 

Method Validation:

The developed technique was proposed to be validated as per ICHQ2 (R1) protocol for analytical method validation to check the validness of the developed method³⁰.

 

Linearity:

Concentration levels of 8, 12, 16, 20 and 24µg/mL were prepared by serially diluting the standard stock solution of Niacinamide and their absorbance were recorded in the five selected wavelength around the λ_max of the drug (262nm), i.e., 266, 264, 262, 260 and 258nm. The overlay UV spectra showing linearity is represented in Figure 3.

 

 

Figure 3: Overlay of UV spectrum showing linearity

 

The absorbance values recorded for the prepared concentrations at five different selected wavelengths were tabulated in Table 1. Linearity data showing system suitability parameters at the selected wavelengths were tabulated in Table 2. The linearity graphs and the residual plots were depicted in Figures 4- 13.

 

Table 1: Multivariate UV calibration at five selected wavelengths

Concentration (µg/ml)	Absorbance (nm)
	266	264	262	260	258
8	0.252	0.281	0.295	0.287	0.277
12	0.319	0.360	0.362	0.359	0.348
16	0.409	0.451	0.472	0.458	0.443
20	0.510	0.560	0.580	0.582	0.550
24	0.575	0.635	0.664	0.646	0.625

 

 

Table 2: Linearity data showing system suitability parameters at the selected wavelengths

Wavelength (nm)	Regression equation	r2	Average of Slope	SD of Intercept	LOD (µg/ml)	LOQ (µg/ml)
266	Y=0.0209x+0.0782	0.994	0.02276	0.00551	0.79	2.42
	Y=0.0230x+0.0876	0.995
	Y=0.0242x+0.0902	0.996
	Y=0.0232x+0.0924	0.996
	Y=0.0225x+0.0894	0.995
264	Y=0.0227x+0.0942	0.996	0.02332	0.0841	0.73	2.22
	Y=0.0236x+0.0958	0.997
	Y=0.0248x+0.0968	0.998
	Y=0.0232x+0.0952	0.998
	Y=0.0223x+0.0841	0.997
262	Y=0.0239x+0.0922	0.994	0.02568	0.00604	0.77	2.35
	Y=0.0245x+0.0988	0.995
	Y=0.0278x+0.0995	0.996
	Y=0.0269x+0.0874	0.994
	Y=0.0254x+0.0868	0.994
260	Y=0.0232x+0.0924	0.994	0.02656	0.0057	0.70	2.13
	Y=0.0255x+0.0988	0.996
	Y=0.0298x+0.0985	0.998
	Y=0.0285x+0.0894	0.997
	Y=0.0258x+0.0858	0.996
258	Y=0.0225x+0.0894	0.995	0.0258	0.0062	0.78	2.38
	Y=0.0245x+0.0978	0.996
	Y=0.0292x+0.0985	0.997
	Y=0.0282x+0.0864	0.996
	Y=0.0248x+0.0858	0.995

 

Limit of Detection (LOD) and Limit of Quantification (LOQ):

Limit of detection (LOD) and Limit of quantification (LOQ) values were calculated employing the slope and intercept values of the linear regression line, thereby the sensitivity of the developed method was established. (Table 2).

 

 

Precision:

Interday and intraday precision studies were performed by measuring the absorbance of 100% concentration level of the linearity solution, i.e., 16µg/mL, three times a day (intraday) and on three different days (interday). The recorded absorbance values for intraday and interday precision studies were provided in Table 3 and 4 respectively. The calculated SD and % RSD values for the precision studies were represented in Tables 5 and 6.

 

Table 3: Absorbance values for intraday precision

 

 

 

Table 4: Absorbance values for Interday precision

 

 

 

 

Table 5: Intraday precision

 

 

 

 

Table 6: Interday precision

 

 

Table 7: Assay of Niacinamide

 

 

Table 8: Recovery studies

Wave length (nm)	Conc. levels (%)	Sample Conc. present (µg/ml)	Standard Conc. added (µg/ml)	Final Conc. (µg/ml)	Amount recovered (µg/ml)	% Recovery
266	50	4	4	8	7.94	99.25
	100	4	12	16	16.11	100.68
	150	4	20	24	23.87	99.45
264	50	4	4	8	8.14	101.75
	100	4	12	16	16.41	102.56
	150	4	20	24	24.20	100.83
262	50	4	4	8	8.17	102.12
	100	4	12	16	15.81	98.81
	150	4	20	24	23.87	99.45
260	50	4	4	8	7.84	98.00
	100	4	12	16	15.74	98.37
	150	4	20	24	24.21	100.87
258	50	4	4	8	8.02	100.25
	100	4	12	16	15.95	99.68
	150	4	20	24	24.04	100.16

 

Assay:

The absorbance of the sample solution was recorded at 262nm and the amount of drug in the selected pharmaceutical dosage form was estimated and reported in Table 7.

 

Accuracy (Recovery studies):

The accuracy of the developed method was evaluated by recovery studies employing standard addition method at 50%, 100% and 150% concentration levels. Three different 10mL volumetric flask was taken in which 0.4mL each of sample stock solution was added into all the three flasks, followed by the addition of 0.4, 1.2 and 2.0mL of standard stock solution respectively and the final volume was made up to the make with distilled water. The absorbance of the above solutions was recorded. The percentage recovery of the drug was calculated and reported in Table 8.

 

RESULTS AND DISCUSSION:

The absorption maximum of Niacinamide was found to be 262nm.

 

Linearity:

The developed method was reported to be linear within the specified concentration range of 8 – 24µg/mL. Linear regression equation was constructed for all the five selected wavelengths of 266, 264, 262, 260 and 258 nm. The correlation coefficient values obtained was found to be more than 0.99.

 

Limit of Detection (LOD) and Limit of Quantification (LOQ):

The LOD and LOQ values calculated were found to be 0.70 – 0.79µg/mL and 2.13 – 2.42µg/mL respectively.

 

Precision:

Intraday and interday precision studies were carried out. Percentage RSD values for intraday and interday precision were observed to be in the range of 0.26- 0.36 and 0.71- 0.88 respectively, which was found well below the acceptance criteria of 2% as per ICH guidelines. The low estimated %RSD value demonstrates that the developed technique was precise.

 

Assay:

The absorbance of the sample solution was recorded at 262nm and the quantity of Niacinamide present in the tablet formulation was calculated. The assay percentage of the drug was found to be 99.91% w/w and the calculated percentage RSD was found to be less than 2%.

 

Recovery:

The percentage recovery of the drug was calculated and was observed to be within the range of 98.00- 102.12% w/w, which was found to be within the limit of 97- 103 % w/w according to ICH protocol. Hence the method can be reported as accurate.

 

CONCLUSION:

The developed simple and rapid UV spectrophotometric aided Multivariate calibration technique was found linear, sensitive, accurate and precise for the evaluation of Niacinamide in tablet formulation. All the validation parameters assessed were reported to be within the limit specified in accordance to ICH guidelines. The developed method can be extended for the quantification of Niacinamide in various other available dosage forms of the drug such as tablet, gel and injectable powders. Therefore, a simple and rapid method using mathematical contents was developed, which was found more predictable than the other spectrophotometric methods, as the absorbance of the drug is measured at five different selected wavelengths, which makes the method more reliable than the currently available official method in IP 2018 (Vol-III). Hence the method is strongly recommended for the routine quality control analysis of Niacinamide in Pharmaceutical formulations.

 

 

ACKNOWLEDGMENT:

Authors are thankful to the Chancellor, SRM Institute of Science and Technology and the Management of SRM College of Pharmacy, SRM Institute of science and Technology, Kattankulathur for providing the facilities required for carrying out this research work successfully.

 

AUTHORS CONTRIBUTION:

All the authors have contributed equally in designing the analysis, for the collection of data, in performing the analysis, and to write the research work in the instructed manner to frame the final manuscript in a successful manner.

 

CONFLICT OF INTEREST:

The authors repot on conflicts of interest on the study.

 

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Received on 13.07.2019            Modified on 28.11.2019

Accepted on 21.04.2020         © RJPT All right reserved