vitamins are essential for nutrition and physiological function , and therefore , their intake is necessary for humans and animals , as the body is unable to synthesize these essential nutrients required for growth . vitamins can be classified into water - soluble and fat - soluble vitamins ; the former includes niacin , nicotinamide , vitamin b6 , vitamin b2 , vitamin b1 , folic acid , pantothenic acid , and vitamin c , while the latter includes vitamin a , vitamin d , vitamin e , and vitamin k . vitamins have specific and important functions , and as such , both humans and animals require vitamins to achieve and maintain health and productivity . vitamins function to control various endogenous metabolic activities taking place in the body , such as energy and amino acid metabolism . although only small amounts of vitamins are required , vitamin deficiency ( or indeed , excess ) can lead to diseases such as beriberi   and dermatitis [ 2 , 6 , 7 ] . thus , to prevent a vitamin deficiency or excess in animals , as well as to attain maximum performance , the control of feed additives is necessary . indeed , vitamins are essential for maintaining normal metabolic processes in animals , while also retaining their condition and performance . as animals are unable to synthesize vitamins , the small amounts of these nutrients required must be supplied in their feed . various methods have been reported for the characterization of water - soluble vitamins , such as high performance liquid chromatography ( hplc ) coupled with ultraviolet ( uv ) detection [ 10 , 11 ] , liquid chromatography ( lc ) coupled with mass spectrometry ( ms ) , lc coupled with ms / ms , and gas chromatography . the most commonly used methods for the determination of vitamin components are based on hplc separation . lc - ms and lc - ms / ms , which provide information about the molecular mass and structural features of components , are considered more useful than other methods for the separation , identification , and quantification of the characteristic vitamin compounds . however , since these methods are expensive to purchase and maintain , many laboratories prefer hplc - uv detection , which is less costly , relatively convenient to operate , and suitable for the routine analysis of vitamins . ion - paring reagent we used in this study has amine group , so it is suitable for water - soluble vitamins analysis as well as iodine speciation analysis and pesticide residue analysis [ 1618 ] . in this study , six water - soluble vitamins in animal feed ( nicotinic acid ( niacin ) , nicotinamide , folic acid , riboflavin , pyridoxine ( vitamin b6 ) , and thiamine ( vitamin b1 ) ) complete animal feeds have complex matrixes containing fat , protein , carbohydrate , salt , and so on . water - soluble vitamins we targeted have amine functional group which represents polarity . to increase the selectivity of the water - soluble vitamins in complicated matrixes , in addition , simultaneous analytical method for the determination of 6 water - soluble vitamins has been rarely reported . moreover , therefore , this newly developed simultaneous method should be validated in various feed matrixes . a novel analytic method is therefore reported , which is suitable for detecting and quantifying the vitamins present in animal feed , while also aiding in the management of feed standards . a total of 30 animal feed samples were used , and 20 dried samples were pulverized into fine powders ( hmf-100 , hanil electric co. , seoul , korea ) . the pulverizer was set at a maximum speed of 22,000  rpm to give fine powders ranging in size from 400 to 1000  m . hplc grade acetonitrile , methanol , and acetic acid were purchased from merck ( darmstadt , germany ) , and picb-6 ( pic , paired - ion chromatography ; b , separate bases ) was purchased from waters ( st . milford , ma , usa ) . water was purified using a milli - q rios/elix water purification system ( millipore , bedford , ma , usa ) . nicotinic acid ( niacin ) , nicotinamide , pyridoxine ( vitamin b6 ) , riboflavin , thiamine ( vitamin b1 ) , and folic acid standards were purchased from sigma aldrich chemie gmbh ( bellefonte , pa , usa ) . the six vitamins were classified into three groups , groups 1 , 2 , and 3 , and stock solutions were prepared using the preprocessing reagent , water , and 0.01  n naoh to obtain a final vitamin concentration of 100 group 1 consisted of thiamine and pyridoxine , and the stock solutions were prepared by mixing the standard preparation with water ( 100  ml ) . group 2 consisted of riboflavin and folic acid and was prepared by mixing the standard preparation ( 10  mg ) , 0.01  n naoh , and water ( 100  ml ) . group 3 consisted of nicotinic acid and nicotinamide and was prepared by mixing the standard preparation ( 10  mg ) with the preprocessing reagent ( 100  ml ) . working solutions were prepared by diluting the stock solutions , and all solutions were refrigerated . the samples were prepared for extraction by the ion - pairing reagent . each homogenized sample from animal feed ( 1.5  g ) was placed into a centrifuge tube and mixed with the extraction solvent ( 10  ml , 5  mm picb-6 in 0.1% ch3cooh ) . a portion of the liquefied sample ( 10  g ) was added to a 25  ml volumetric flask , and the volume was made up to 25  ml using the extraction solvent ( 5  mm picb-6 in 0.1% ch3cooh ) . we used picb-6 as ion - pairing reagent because most of vitamins we determined showed higher resolution when we used picb-6 than other ion - pairing reagents . the mixtures were then homogenized for 10  min , sonicated for 10  min at room temperature , and centrifuged at 13,000  rpm for 10  min at 4c . the resulting supernatant was filtered through a 0.45  m disposable filter ( whatman ) . finally , the stabilities of the standard preparations were determined over one week in an amber vial at room temperature , with samples being taken for analysis every 24  h. hplc was performed on a shiseido nanospace si-2 system ( shiseido , tokyo , japan ) equipped with a binary solvent delivery pump , an autosampler , and a photodiode array detector ( pda ) and was controlled using the ezchrome elite software ( agilent technologies , palo alto , ca , usa ) . a reversed phase unison uk - c18 ( 100  m  4.6  mm , 3  m particle size ) ( tokyo , japan ) column was used for all separations at a column temperature of 40c . prior to use , the mobile phase was filtered through a 0.45  m membrane filter ( millipore , milford , ma , usa ) and degassed under vacuum . the mobile phase was composed of buffers a ( 5  mm picb-6 in 0.1% ch3cooh ) and b ( 5  mm picb-6 in 65% methanol ) with the following gradient elution : 0  min , 10% b ; 02  min , 10% b ; 222  min , 70% b ; 2227  min , 70% b ; 27 - 28  min , 10% b ; and 2835  min , 10% b. the sample injection volume was 2  l , and the flow rate was set at 0.5  ml / min . method validation was performed according to the guidelines set by the international conference on harmonization ( ich , 2005 )   and the international union of pure and applied chemistry ( iupac , 2002 ) . the method was validated for linearity , sensitivity , selectivity , accuracy , and precision , as outlined in figure 2 and table 1 . the developed rp - hplc / uv method incorporating an ion - pairing reagent was validated to verify that its performance met the requirements for routine vitamin analysis . several performance characteristics were measured , including selectivity , linearity , sensitivity , accuracy , and precision . selectivity was determined by the absence of interference in the chromatographic window , and this was measured using blank chromatograms at the specific quantification wavelength ( i.e. , 270  nm ) . as shown in figure 2 and table 1 , the chromatograms of the water - soluble vitamins indicate successful separation of all six compounds in < 29  min , with good resolution ( 5.5534.85 ) and asymmetry ( 0.941.08 ) , thereby indicating satisfactory selectivity for this hplc system . linearity was assessed by building external calibration curves for each compound using the vitamin - containing working solutions . calibration curves were obtained by plotting the analyte peak area versus its concentration over seven different concentrations . each concentration of the mixed standard solution was injected in triplicate , and then , the regression parameters were calculated . the sensitivity of the developed method was evaluated by determining the lod and loq values . under the described chromatographic conditions , these values were calculated based on the response and slope of each regression equation at signal - to - noise ratios ( s / n ) of 3  :  1 and 10  :  1 , respectively . for the different components , the lod values ranged from 25 to 197  g / kg , while the loq values ranged from 84 to 658  g / kg . the method precision was then determined by measuring the intra- and interday precision . for the intraday precision , six replicates of the mixed standard solutions were analyzed within 1  d , while the solutions were examined in triplicate for three consecutive days for the intraday precision . the precision was expressed as the percentage of the relative standard deviation ( % rsd ) . the overall intraday % rsd values were < 5.6% , while the interday values were < 4.8% ( see table 1 ) . the accuracy was evaluated by adding the mixed standard solutions at two different concentrations ( high : 20.0  mg / kg ; low : 2.0  mg / kg ) to feed from the association of american feed control official ( affco , 201591-swine mineral and vitamin supplement ) . the mixture was extracted using the developed ion - pairing reagent technique and hplc method . excellent recovery rates of 82.398.9% indicated a high level of accuracy for this method , as detailed in table 1 . the stability of all standard solutions was also tested over 7  d , with the majority having decomposed within 3  d. in particular , in the case of folic acid , rapid decomposition was observed after 3  d compared to that of the other water - soluble vitamins ( i.e. , 63.38% cf . these results demonstrated that it is necessary to prepare a fresh standard preparation for each analysis . based on the above validation data , the proposed method was concluded to provide good linearity , sensitivity , selectivity , accuracy , and precision for the simultaneous analysis of water - soluble vitamins . among the 30 different animal feeds that were analyzed in this study , vitamins were detected in all animal feeds purchased from supermarkets ( table 3 ) . identification of the six compounds was by comparison of their retention times and uv spectra with those of the standards and the pure compounds . the qualitative and quantitative compositions of the six compounds in the animal feed varied significantly . more specifically , the nicotinic acid content ranged from 1,868 to 46,289  mg / kg with an average content of 15,969  mg / kg , while the nicotinamide content ranged from 1,339 to 49,920  mg / kg with an average of 16,257  mg / kg . in addition , the content of folic acid varied between 102 and 5,012  mg / kg ( average = 813  mg / kg ) , while that of pyridoxine was between 2,065 and 49,530  mg / kg ( average = 17,758  mg / kg ) . finally , the riboflavin and thiamine contents ranged from 3,015 to 36,820  mg / kg ( average = 13,286  mg / kg ) and from 1,359 to 48,930  mg / kg ( average = 11,786  mg / kg ) , respectively . thus , a simple , qualitative , and quantitative method for the simultaneous detection and quantification of six vitamin compounds from animal feed was successfully developed and validated using rp - hplc / uv detection . the proposed method showed accuracy and precision and was successfully employed for analyzing different types of feeds . analytical results demonstrated that this hplc method provides a good alternative for routine analysis , owing to its simplicity , specificity , and sensitivity . finally , it exhibits the potential to be applied as a reliable quality evaluation method for animal feed .