food additives are widely used in foodstuffs to prevent from spoilage and improve color , flavor , and texture of foods . however
, these additives in foods may affect individuals who are sensitive with some type of allergy , asthma , and hay fever .
consequently , authorities have set threshold values for acceptable daily intake , varying from country to country .
for instance , the list of authorised food additives and maximum permitted levels in european union are laid down in the annexes of council directive [ 1 , 2 ] . to ensure food safety from farm to fork ,
it is also essential to develop effective and reliable analytical methods for the monitoring of the additive levels in food .
therefore , various analytical methods have been reported for the simultaneous determination synthetic food additives , such as thin layer chromatography , uv - visible spectrophotometry [ 5 , 6 ] , voltammetry [ 7 , 8 ] , differential pulse polarography , capillary electrophoresis , hplc - dad [ 1114 ] , hplc - ms , and hplc - ms - ms [ 16 , 17 ] . until now , although many analytical techniques have been developed for the determination of various food additives in foods , there is no report about simultaneous determination of this combination in food samples . among these analytical methods ,
hplc coupled with uv / vis or diode array detectors ( dads ) are the most commonly used methods due to their sensitivity , selectivity , and high resolution .
so , development of effective chromatographic separation method involves judicious selection of experimental conditions that is suitable for the separation of interested components at an adequate resolution with reasonable run time . in this
it not only is a timesaving method but also it has an ability to reveal possible interactions between variables [ 19 , 20 ] .
hence , experimental designs have been increasingly used to determine the optimum conditions of chromatographic separation of some analytes in food , drug , and biological fluid samples with a minimum number of experiments for over the past decade [ 2128 ] . in this paper ,
a new rp - hplc method was developed , using experimental design , for simultaneous determination of five synthetic food additives in soft drinks , including three synthetic colorants ( carmoisine , allura red , and ponceau 4r ) , two preservatives ( potassium sorbate and sodium benzoate ) , and caffeine . for the optimization procedure , box - behnken design ( bbd ) was used to construct mathematical models that predict how changes input or controlled by variables ( ph , flow rate , and mobile phase ratio ) affected the resolution in defined experimental region .
further , the method validation has been carried out according to the international conference on harmonization guidelines .
the optimized and validated method was successfully applied to some commercial soft drinks containing potassium sorbate , sodium benzoate , carmoisine , allura red , ponceau 4r , and caffeine .
chromatographic analyses were performed using a shimadzu hplc system ( kyoto , japan ) consisting of a model lc20 at pump unit , spd-20a uv - vis detector , 7725  20  l sample injection , a computer , and an inertsil octadecylsilane- ( ods- ) 3v column ( 5  m , 250  mm  4.6  mm ; gl sciences , tokyo , japan ) . the statistical analysis for the analytical responses and validation data
, va , usa ) was used for the graph plotting and for estimating the responses of experimental variables .
milli - q water was used to prepare the solutions and mobile phases ( millipore , milford , ma , usa ) .
sodium acetate trihydrate , glacial acetic acid , and hplc - grade acetonitrile were acquired from merck ( darmstadt , germany ) .
potassium sorbate ( 99.0% purity ) , sodium benzoate ( 99.0% , purity ) , carmoisine ( 98.0% purity ) , allura red ( 98.0% purity ) , ponceau 4r ( 99.0% purity ) , and caffeine ( 100.0% purity ) were purchased from sigma - aldrich ( st . louis , missouri , usa ) .
standard stock solutions of potassium sorbate , sodium benzoate , and caffeine were prepared at a concentration of 250  g / ml .
standard stock solutions of carmoisine , allura red , and ponceau 4r were prepared at a concentration of 100  g / ml . fresh working solutions in the concentration range of 210  g / ml for carmoisine , allura red , and ponceau 4r and 525  g / ml for caffeine , potassium sorbate , and sodium benzoate were prepared by the dilution of the standard stock solutions in milli - q water .
soft drink samples were purchased from local supermarkets in istanbul , turkey , and were degassed in an ultrasonic bath for 5  min .
then , 1  ml of the sample was transferred to a 10  ml volumetric flask and diluted to the volume with milli - q water .
prior to the analysis , both soft drink samples and standard solutions were filtered through 0.45  m millipore filters and then injected into hplc system .
the optimum separation of all analytes was achieved with 0.025  m sodium acetate / acetic acid buffer , ph 6.0 , acetonitrile gradient that follows 05  min , 95  :  75 ( v / v ) ; 510  min , 70  :  30 ( v / v ) .
the mobile phase flow rate was 1.0  ml / min and the injection volume was 20  l in all the chromatographic runs .
the detection was made with a variable ultraviolet - visible detector fixed at 230  nm .
a box - behnken design ( bbd ) using three variables at three levels ( coded levels : 1 , 0 , and + 1 ) was used for the optimization of simultaneous determination of potassium sorbate , sodium benzoate , carmoisine , allura red , ponceau 4r , and caffeine by hplc .
the variables and levels selected for optimization procedure were ph ( a ; 6.0 , 6.5 , and 7.0 ) , flow rate ( b ; 1.0 , 1.2 , and 1.4 ) , and mobile phase ratio ( in terms of acetate buffer ) ( c ; 85 , 90 , and 95 ) ( table 1 ) .
while the first step has an effect on the chromatographic separation , the second step has an effect on the run time of the method .
therefore , experimental variables of the first step of hplc method were taken into account .
15 experimental runs were performed at random and overall resolution ( r ) was chosen as the response for the separation of the compounds . experimental design matrix used and the results obtained by bbd were listed in table 2 . in - house validation of the method
evaluated parameters are linearity of calibration curve , limit of detection ( lod ) , limit of quantification ( loq ) , and precision , accuracy , and stability .
the linearity of the hplc method for the determination of five food additives and caffeine was evaluated in a concentration range of 210  g / ml for carmoisine , allura red , and ponceau 4r and 525  g / ml for potassium sorbate , sodium benzoate , and caffeine covering the normal range of concentrations obtained when analyzing soft drinks .
calibration equations were calculated by the least squares treatment of the peak area of the food additives and caffeine .
the limit of detection ( lod ) and limit of quantitation ( loq ) were calculated as lod 3x/s and loq 10x/s , where  is the standard deviation of intercept and s is the slope . in order to test the prediction performance of the proposed methods , intraday ( three times in a day operation under the same conditions ) and interday ( four different days ) studies were performed at three different concentrations ( level 1 : 10  g / ml ; level 2 : 15  g / ml ; level 3 : 20  g / ml for potassium sorbate , sodium benzoate , and caffeine ; level 1 : 4  g / ml ; level 2 : 6  g / ml ; level 3 : 8  g / ml for carmoisine , allura red , and ponceau 4r ) .
accuracy of the method was ascertained by a recovery study by adding a known amount of reference standards to the soft drink samples .
firstly , 0.5  ml of the soft drink sample was transferred to a 10  ml volumetric flask and the reference standards were added on it at three different concentration levels . then , added samples were diluted to the volume with milli - q water , filtered , and analyzed .
chromatographic optimization requires selecting suitable criteria for the evaluation of the resultant chromatograms in order to choose the optimum conditions .
bbd is an independent , rotatable , or nearly rotatable second - order design based on three - level incomplete factorial designs .
it is more efficient compared to other response surface designs , such as central composite designs .
it can also provide sufficient information to test the lack of fit , and therefore it is one of the best quadratic models for response surface method and has been widely used in analytical fields . because of the nonlinearity of the model , a polynomial function to contain second - order model is postulated to describe the evolution phenomenon:(1)yi = b0+i=1nbixi+i=1nbiixi2+1ijnbijxixj+i , where n is the number of variables , b 
 0 is the constant term , b 
 i , b 
 ii , and b 
 ij represent the coefficient of the first - order terms , quadratic terms , and interaction terms , respectively , and  
 i is a term that represents other sources of variability not accounted for the estimation , such as background noise . the experimental results are shown in table 2 .
the regression model for the response was tested through analysis of variance ( anova ) . from the results of anova ( table 3 )
, it can be deduced that linear contribution of mobile phase ratio ( c ) and quadratic contribution of mobile phase ratio ( cc ) influence the resolution significantly .
interactions of the individual variables in this study are not significant to resolution in the selected range . fitted quadratic model equation is also presented in ( 2 ) .  
figure 1 shows the analysis of individual variables of experimental design . from figure 1 , it can be seen how the value of the resolution may increase if we take higher mobile phase ratio ( c ) .
also , we can infer that although ph ( a ) and flow rate ( b ) do not greatly influence the resolution better resolutions are obtained for low values of ph and flow rate(2)r^=0.67172.99a153.92b+1089.10c+152.74a2345.80ac152.83b2307.58bc+1088.49c2 . 
 
the regression models obtained were used to calculate the response surface for each variable separately .  
figure 2 illustrates the response surface plots for the resolutions . in particular , the effect of ph ( a ) and mobile phase ratio ( c ) on resolution is shown in figure 2(b ) .
this plot shows that the highest resolution is obtained at greater values of the mobile phase ratio .
the relation between the effects of the other variables on the resolution is also plotted in figures 2(a)2(c ) . according to the results of the optimization procedure , the optimum variables corresponded to ph , 6.0 ; flow rate , 1.0  ml / min ; mobile phase ratio , 95% . a typical chromatogram obtained under optimum conditions
the results of the linearity , lods , and loqs are summarized in table 4 .
a good linear relationship is displayed between the corresponding peak areas and the concentrations of the compounds based on the correlation coefficients ( r 
  > 0.9962 ) .
the lods of the six compounds were in the range of 0.100.19  g / ml , and the loqs of the six compounds were in the range of 0.330.63  g / ml .
a summary of intraday ( the rsd of the recoveries of the nine samples ) and interday precision ( the rsd of the recoveries of the twelve samples ) are listed in table 5 .
these results show that the proposed method is precise for the simultaneous determination of these compounds .
the recoveries of the six compounds to determine the accuracy of the method are summarized in table 6 .
the proposed method resulted in satisfactory recoveries for all additives and caffeine , ranging from 97.67% to 105.56% .
the recoveries demonstrated that the matrixes have negligible effect on the quantification of these compounds and the method is accurate within the desired range . under refrigerated and room temperature conditions , all food additives and caffeine in mobile phase and water were stable for at least 1 month .
these results show that the proposed method is precise , accurate , and sensitive for the simultaneous determination of the six compounds and can be used for routine analysis of potassium sorbate , sodium benzoate , carmoisine , allura red , ponceau 4r , and caffeine in soft drinks .
the proposed hplc method was applied to the simultaneous determination of potassium sorbate , sodium benzoate , carmoisine , allura red , ponceau 4r , and caffeine in different soft drinks .
five replicates determination was made and the results are summarized in table 7 . the concentration of food additives in soft drinks ranged from 24.26  0.47  g / ml to 254.13  1.24  g / ml . the amounts of food additives and caffeine in all soft drink samples were below the limit value defined in the legislation on the food additives [ 1 , 2 ] .
an efficient , accurate , and reliable method for the simultaneous determination of five food additives and caffeine in soft drinks was developed using hplc .
box - behnken design was applied to the optimization of the chromatographic separation conditions and this design reduced to the number of experiments required .
it can be concluded that a slight change in mobile phase ratio has a direct effect on the resolution .
high percentage recovery data also shows that the proposed method is free from the interference .
consequently , this study will provide a sensitive and rapid method for the detection of potassium sorbate , sodium benzoate , carmoisine , allura red , ponceau 4r , and caffeine in soft drinks .
 box - behnken design was applied to optimize high performance liquid chromatography ( hplc ) conditions for the simultaneous determination of potassium sorbate , sodium benzoate , carmoisine , allura red , ponceau 4r , and caffeine in commercial soft drinks . 
 the experimental variables chosen were ph ( 6.07.0 ) , flow rate ( 1.01.4  ml / min ) , and mobile phase ratio ( 8595% acetate buffer ) . 
 resolution values of all peak pairs were used as a response . 
 stationary phase was inertsil octadecylsilane- ( ods- ) 3v reverse phase column ( 250  4.6  mm , 5  m ) dimensions . 
 the detection was performed at 230  nm . 
 optimal values were found 6.0 ph , 1.0  ml / min flow rate , and 95% mobile phase ratio for the method which was validated by calculating the linearity ( r 
 2 > 0.9962 ) , accuracy ( recoveries  95.75% ) , precision ( intraday variation  1.923% , interday variation  1.950% ) , limits of detection ( lods ) , and limits of quantification ( loqs ) parameters . 
 lods and loqs for analytes were in the range of 0.100.19  g / ml and 0.330.63  g / ml , respectively . 
 the proposed method was applied successfully for the simultaneous determination of the mixtures of five food additives and caffeine in soft drinks . 
1. Introduction
2. Experimental
3. Results and Discussion
4. Conclusion