Determination of Water-Soluble Vitamins in Iraqi Honey Bee and Compare with Others Types by High – Performance Liquid Chromatography

High-performance liquid chromatographic methods are used for the determination of water-soluble vitamins with UV-Vis. Detector. A reversed-phase high-performance liquid chromatographic has been developed for determination of water-soluble vitamins. Identification of compounds was achieved by comparing their retention times and UV spectra with those of standards solution. Separation was performed on a C18 column, using an isocratic 30% (v/v) acetonitril in dionozed water as mobile phase at pH 3.5 and flow rate 1.0m/min. The method provides low detection and quantification limits, good linearity in a large concentration interval and good precision. The detection limits ranged from 0.01 to 0.025μg/ml. The accuracy of the method was tested by measuring average recovery values ranged between 94% 101 %. For standerd solution, and 93%-99% of honey bee samples.


Introduction
As generally known, vitamins are essential substances, which are necessary for normal health and growth and in sufficient amounts should be supplied by food.If this intake is insufficient or if special dietary requirements exist, multivitamin preparation should be taken in order to prevent vitamin deficiency.Honey is a natural substance produced by bees and is a nutritious food of economic Importance worldwide [1][2][3].Honey is a sweet and viscous fluid created by honeybees from the nectar of flowers [4].Honey is at most composed of a complex mixture of carbohydrates and other minor substances, such as organic acids, amino acids, proteins, minerals, and vitamins.In roughly all honey types, fructose predominates glucose being the second main sugar.These two account for nearly 85-95% of the honey carbohydrates.More complex sugars made up of two or more molecules of glucose and fructose constitute the residual carbohydrates, except for a trace of polysaccharide.Honey also consists of volatile substances which are responsible for the characteristic aroma [5,6].Vitamins are organic substances that are essential in amounts increase activity of the body.They are naturally source from plant and animal foods.The amounts of vitamins ingested from food are measured in micrograms or milligrams [7].Eight of the watersoluble vitamins are known as the vitamin B-complex group: riboflavin (vitamin B2), niacin (vitamin B3), thiamin (vitamin B1), folate (folic acid), vitamin B6 (pyridoxine), Riboflavin Vitamin B12, biotin and pantothenic acid.The B vitamins are distributed in foods .Water-soluble vitamins are not stored in the body and are easily excreted while fat-soluble vitamins are cumulative in the body and the extravagant assimilation of vitamins A (Retinol) and D (Calciferol) can prove to be harmful [8][9][10].

Chemicals
All the used chemicals were of the highest purity available analytical grade.Deionized water is used for all purposes.All glass ware, tubes, volumetric flasks, pipettes, tips and other glass were immersed in HNO 3 (5% V/V) for 24 hr.then, rinsed with deionized water.All solvents in this study were grad-HPLC and obtained from BDH, Supelco company (USA) and sigma Company.

Preparation stock Solutions of vitamins
The mixture of soluble vitamins was prepared by dissolving (Thiamine.

Extraction Samples
One gm of homogenized honey were weighed and dissolved in 1.0 ml of ultra-pure water.Then, 0.1 ml of 2M NaOH diluted (in order to favor the complete solubilization of the honey), and the solution was topped up to mark with ultra pure water in a 25 ml volumetric flask.Sample solutions were injected through a PVDF (13 mm and 0.45 m) .The honey solution was stored in the dark at 4 •C until injection [11].

Results and Discussion
Vitamins were essential for human healths which are classified into two groups dissolved in water and soluble in fat.All water soluble vitamins are not stored in the body except B12and B6.These vitamins play an important role in vital functions such as metabolism [12].Honey consists of a mixture of complex such as compounds(flavonoids and phenolic acids, amino acid) which shows various absorption in the UV region compounds, so it is a separation method are most suitable for the extraction of these compounds.We could do by using HPLC method [13].

Optimization of the Separation of vitamins
The Effect of pH on the retention time The effect of mobile phase pH on in reversed phase HPLC for separation mixture vitamins, to observe the effect of various pH on the retention time of standards.The pH of the system was then varied between ranges of (2.5-7.5), it was adjusted by a few drops of either (0.1 M)HCl or (0.1 M) NaOH solution .The effect of pH changes on the retention time of vitamins show in Table (3).A plot of adjusted retention time (tR) for vitamins , versus pH were present in Figure (1).The optimum pH obtained for best baseline separation of vitamins pH 3.5.However, it was not possible to cover pH range less than 2.0 and more than 8 due to instability of the packing over this region since the alkaline solution dissolves the silica support and at low pH breaks the Si-O linkage.While at pH region between 5.0-7.0 the retention time is highly changed by the pH variation.This can be attributed to less effective ion-pairing reagent at the higher pH.The pH 3.5 was selected at the best one for vitamins.

Optimization of concentration of Mobile Phase in on elution of water soluble vitamin
In reveres phase methods water is one of the solvents used in the process of and it does not compete with the analyte for the adsorption sites.Washing the column another component of (e.g.acetonitrile), so that usually a modifier because it can interact with the adsorbent surface and compete with analyte molecules for the adsorption sites for dissolve this problem by Increasing the concentration of the modifier in the eluent leads to the decreasing of the analyte retention time.This study was done by using deionized water acidified with and acetonitrile in different ratios.This stage was done by the system of HPLC.The results show generally that the retention time was decreased with increasing the % of acetonitrile.This effect has been attributed to a decrease of the surface concentration of the counter-molecule because of the competition by the solvent.

Calibration curve
The optimum condition for the separation of amino acid and water soluble vitamin were tabulated in Table (6

Resolution Measurement
Column performance traditionally has been defined by its reproducibility.Height equivalent to the theoretical plates, HETP (H) and number of plates although useful., but these parameters do not provide sufficient information for properly evaluating column usefulness, capacity factor K was also recommended for column evaluation [14] .The results listed in Table (10) show the column parameters for an optimum separation of water soluble vitamins The partition ratio K , which is commonly called the capacity factor.The capacity factor K for water soluble vitamin chromatographic on ODS column were ranged (2.9-8.265) for the analyzed samples as listed in Table (10).The column selectivity, originally called the separation factor  is defined as the ratio of the capacity factors of two adjacent peaks = 2 K / 1 K ) The  represents the separation factor or selectivity representing capacity factor between the two components  values for water soluble vitamins (1.2-1.4) as listed in Table (10) When the value of  1 or more, that means we obtained base line separation for all the eluted mixture.
Therefor the optimum separation condition we got, gave mixture under this study, The phase separation mechanism in ODS column depends on the hydrophobic binding interaction between the R group of organic compounds and immobilized hydrophobic ligand of the stationary phase .The different values of retention times due to interaction between different R groups of organic compound and octadecyle group of the stationary phase governed the mechanism of the retention time.This decreased hydrophilic or hydrophobic interaction with the stationary [14].

Application of the Optimum Conditions for the separation and determination of Water Soluble Vitamin
The analysis of water soluble vitamins in variation honey samples was applied under the optimum condition.The optimum conditions for the separation of water soluble vitamins were obtained by variation in the organic modifier, pH and flow rate.The experimental results of these studies observed the optimum conditions, which gave a base line separation for the whole mixture 30% acetontrile water soluble vitamins respectively , pH 3.5 and flow rate 1ml /min for ,as shown in typical chromatogram Figures (5) [15] and B6 in Brazilin honey 4074 mg/kg [16].

Conclusions
This study was focused to determine Water -soluble vitamins in Iraqi honey samples that produce in University of Baghdad College of Sciences for Women and other located and compared with other kinds.The obtained results found that it is possible to take advantage of this method that was developed in the set and extract the active compounds in the honey which is water-soluble vitamins of the importance vital.The extraction and separation using a HPLC technique high response, sensitivity and speed too high in separation.Close results and agreement of Iraqi honey samples when compared by extraction methods for honey samples with literature.
Fig. (3): Retention time as a function of flow rate for Water soluble vitamins.

Table ( 4): Variation of retention time (tR) of water soluble vitamins on reversed phase at different % acetonitrile flow rate 1 .0ml/min
Table (4) shows variation of retention time of water soluble vitamins