INTRODUCTION:

Vitamins plays a key role in performing various chemical and physiological functions in body. These vitamins are important nutrient and should be taken in small amounts regularly. Based on their solubility, they are categorized into two groups: Water soluble vitamins and Fat soluble vitamins [1]. Water soluble vitamins plays a very important role due to its reducing and anti oxidative properties as well as co-enzymatic functions for the metabolism of other nutrients. Both Biotin (vitamin B7) and Folic acid (vitamin B9) are B Group vitamins [2].

Biotin participates in numerous metabolic reactions because of its critical role as a cofactor for multiple carboxylase enzymes [3]. It is an important component of enzymes in the body that break down certain substances like fats, carbohydrates, and others.

It is used to treat symptoms of biotin deficiency associated with pregnancy, malnutrition, and long-term tube feeding. It is also used orally for treating hair loss, brittle nails, skin rash in children, mild depression, and abnormal glucose levels in the blood caused by reduced biotin levels in the body [4] [5].

Folic acid occurs naturally in food, but it is not suitable for fortified foods or supplements [4]. The derived form of folate is folic acid that can be added in supplements and foods due to its increased stability compared to its natural form and it helps the body to produce and maintain new cells, and also helps prevent changes to DNA that may lead to cancer. It is sometimes used in combination with other medications to treat pernicious, aplastic, or normocytic anaemia. Biotin and folic acid are commonly prescribed for Pregnant women and Anaemic patients [4-7].

As per the Literature survey, no particular method was developed yet, though the formulation was available in the market, only few chromatographic and spectrophotometric methods has been reported for estimation of Biotin and Folic acid separately not in a simultaneous method [7-11]. Hence, the aim of this study is to develop and validate a new chromatographic simultaneous method of analysis for Biotin and Folic acid in Bulk and dosage form.

MATERIALS AND METHODS:

The Reference standard of Biotin and Folic acid were purchased from Fourrts Laboratories, Chennai. The commercial product of Biotin and Folic acid was bought from market separately. HPLC grade Methanol was purchased from Sigma Aldrich Chemicals Pvt. Ltd, Mumbai. Ammonia solution was purchased from Thermo fischer Pvt Ltd. 0.1 N NaOH was obtained from LOBA Chemicals Pvt. Ltd. Ultra pure water was obtained from Mille Q-water purification system from Millipore (miliford) USA.

Instrumentation:

HPLC system (Shimadzu, LC-20AR series, CDM 20A), Shimadzu-SPD-M20A photo diode array detector, C18 Sun fire (250x4.6mm I.D,5μmparticle size) column, UV-1650PC (Shimadzu, UV probe) were used in this study.

Methods:

Preparation of buffer solution:

0.05M Potassium dihydrogen orthophosphate buffer was prepared by dissolving 6.8g of Potassium dihydrogen buffer in 1000ml of water and pH was checked with digital pH meter.

Preparation of Standard solution:

Weigh 10 mg of Biotin and 10mg of Folic acid separately and dissolved in 10ml of 0.1 N NAOH. From this Stock solution ,1-5ml was taken and appropriately diluted with mobile phase. Stock calibration curve were prepared from 10-50μg/ml for Biotin and 10-50μg/ml. Similar procedure was followed for mixtures.

Preparation of Sample solution:

Weigh and powder 10 tablets. Transfer an accurately weighed quantity of finely powdered tablets equivalent to 5 mg of Biotin and 5mg of Folic acid were taken in 10 ml volumetric flask, add about 10ml of 0.1M NaOH and sonicate for 15 min, then filter it with Whatmann filter paper (No. 41) and it was diluted to volume with mobile phase with the concentration range of 10-50μg/ml.

RESULTS AND DISCUSSION:

Selection of analytical wavelength:

About 10mg of reference standard of Biotin were dissolved in 10ml of 0.1N NaOH to yield a stock solution of 1000μg/ml. From this 1ml was taken and make up to 10ml with dilute ammonia as a secondary stock of 100μg/ml. Then, 0.2ml-1ml of secondary stock solution was taken and diluted to 10ml with dilute ammonia to yield dilution of 2-10μg/ml. Similarly, stock solution for folic acid was prepared to yield 2-10μg/ml. Standard solutions were scanned in the range of 200-400nm and then both spectra was overlapped to each other and it will show the point at higher absorbance of both drugs occurs. The UV overlap spectra so obtained shows that the wavelength of isobestic point is at 212nm, which was selected as a working wavelength for the simultaneous analysis. (Fig.1).

Fig. 1: UV overlain spectrum of Biotin and folic acid

OPTIMIZATION OF CHROMATOGRAPHIC CONDITION:

Different mixtures of organic solvents, aqueous phase and phosphate buffer combinations were tried with standard working solution of 10-50μg/ml. Hence ideal mobile phase for the study was found to be 0.05M Potassium dihyrogen orthophosphate buffer: Methanol (70:30 v/v). These mobile phase gives a sharp peak and Stationary phase of Sunfire C₁₈ column (250х4.6mm I.D,5μm particle size) was used. Mobile phase pH was adjusted between 3.0-9.0 with orthophosphoric acid and NaOH but mobile phase pH of 5.3 gives good peak with better resolution. Flow rates of 0.5ml/min,0.6ml/min and 0.7ml/min were tried, 0.6ml/min was used for the study. Final optimized chromatographic condition were shown in table 1.

Table 1: Final optimized condition

S. No.	Parameters Specification
1.	Stationary phase: Sunfire ^TMC₁₈ (250 x 4.6 mm),5μm
2.	Mobile phase
3.	Solvent ratio: 70:30 v/v
4.	pH: 5.3
5.	Detection Wavelength: 212nm (Isobestic point)
6.	Flow Rate: 0.6 ml/min (Isocratic run)
7.	Sample Volume: 20μl
8.	Temperature: 25˚ C

Chromatogram shows the separation of both the drugs in mix standard. Retention time was found to be 6.4 min for Biotin and 3.8 min for Folic acid. (Fig. 2). Estimates obtained from the formulation was found to be within the limits as per ICH Guidelines in table 2.

Fig 2: Chromatogram of mixture (Biotin and Folic acid)

Table 2: Analysis of Formulation

S. No	Drug	Amount(mg)		% Label claim	% RSD
S. No	Drug	Labelled	Found	% Label claim	% RSD
1.	Biotin	5	4.4	88%	0.74
2.	Folic acid	5	4.56	91%	0.85

The developed analytical simultaneous method was validated as per ICH Q₂R₁Guidelines [12]. Parameters such as Linearity, Precision, Accuracy, LOD, LOQ, Specificity, Robustness and System suitability parameters were taken up as test for analytical method validation.

Linearity:

The developed method shows good linearity with the concentration range of 10-50μg/ml for both Biotin (Fig. 3) and Folic acid (Fig 4). A Calibration graph was plotted by taking concentration on X axis and peak area on Y axis which gave a straight line with slope of y=10201x+1171.3 for Biotin and y=92905x+28936 for Folic acid.

Fig:3: Linearity graph of Biotin

Fig: 4: Linearity graph of Folic acid

Precision:

Precision was determined by measuring a minimum of three different concentrations. It was of three types Intraday precision (three different injections at three different times in the same day), Interday precision (three different injections at three different days over a period of a week) and Repeatability (injecting same concentrations for five times). %RSD were calculated. Results were shown in the table 4.

Accuracy:

Accuracy was carried out by the assessement of difference between the assisted value and real value. Three different concentrations of 10μg/ml, 30μg/ml and 50μg/ml at three different levels of 80%, 100% and 120% were selected. Mean % Recoveries were reported in the table 4.

Limit of Detection and Limit of Quantification:

LOD and LOQ were determined by measuring the lowest amount of analyte that can be detected and quantifed respectively. Results were shown in the table 4.

Specificity:

Selectivity is the ability of an analytical method to differentiate and quantify the analyte in presence of other components in the sample. Selectivity is evaluated by injecting the blank and comparing with the response of extracted LLOQ samples processed with NaOH solution and Mobile Phase. Endogenous interferences were not detected at the retention time of selected drugs and standard. These observations show that the developed assay method is specific and selective.

System suitability parameters:

System suitability of the method was performed by calculating the chromatographic parameters like Column efficiency (theoretical plates), Resolution factor, Peak asymmetric factor, Tailing factor were measured and calculated by repetitive injection using system suitability test solution. Results have been reported in the table 3.

Table 3: System suitability parameters

S. No	System suitability parameters	Biotin	Folic acid
1.	Tailing factor	1.01	1.55
	Theoretical plates	5324	4658
	Asymmetry factor	1.2	1.0
	Resolution	2.6

Robustness:

Robustness of the developed method were determined by changing the flow rate ±0.1ml/min (0.5ml/min and 0.7ml/min), changing the mobile phase pH by ±0.2% (5.1 and 5.5) and changing the detection wavelength by ±2nm (210nm and 214nm). It was observed that no marked changes in the chromatogram so the method was robust in nature.

Table 4: Validation parameters summary.

S. No.	Parameters	Observations
1.	Linearity:
	Biotin	0.996
	Folic Acid	0.999
2.	Accuracy:
	Biotin	100.5 %
	Folic Acid	99.81 %
3.	Precision:	Intraday	Interday	Repeatability
	Biotin	0.8	0.81	1.43
	Folic Acid	0.9	0.83	1.2
4.	LOD
	Biotin	0.07
	Folic acid	0.03
5.	LOQ
	Biotin	0.2
	Folic acid	0.9

The working condition for the RP-HPLC method were established for Biotin and Folic acid then applied on Pharmaceutical dosage form. Retention time was found to be 6.4 min for Biotin and 3.8min for Folic acid with the better resolution of 2.6. The Correlation Coefficient (r²) for both drugs was greater than 0.996 (10-50μg/ml). Recovery was carried at three levels (80%,100%,120%) and the results shows that this method is capable of recovering analytes from its formulation. For all the validation parameters %RSD was found to be < 2 which was within limits as per ICH Guidelines.

CONCLUSION:

An Isocratic method has been developed for the simultaneous estimation of Biotin and folic acid in Bulk and Tablet dosage form by RP-HPLC. The Proposed method was simple, rapid, economic, less time consuming when compared to other methods because peaks are eluted within 10 min, so it offers a significant advantage of short run time. Parameters such as Linearity, Precision, Accuracy, LOD, LOQ and system suitability was validated as per ICH guidelines and the results show that the method was reliable and acceptable. Therefore, we conclude that the method was suitable for application in routine analysis of Pharmaceutical preparation. The final optimized condition was also suitable for the further studies by using Plasma.

REFERENCES:

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Received on 18.10.2019 Modified on 03.12.2019

Research J. Pharm. and Tech. 2020; 13(11):5289-5292.

DOI: 10.5958/0974-360X.2020.00925.7