ferulic acid ( fa ; c10h10o4 ; mw : 194.18  da ) or ( e)-3-(4-hydroxy-3-methoxy - phenyl)prop-2-enoic acid ( figure 1 ) is a chemical compound widely found in vegetables which has a low degree of toxicity after oral administration . in nature , it is biosynthesized from phenylalanine and l - tyrosine by shikimate pathway and occurs mainly as trans isomer [ 2 , 3 ] . fa is characterized as an off - white to light yellowish brown crystalline powder that has aqueous solubility of 6.63  mg / dl at ph 7.2 , pka of 4.58 , melting range between 168 and 171c , half - life of 42  min , and log  p of 1.67 . in particular , fa has been proposed as a novel antioxidant drug endowed with a strong cytoprotective activity due to both the ability to scavenge free radicals and activate cell stress response . previous studies have demonstrated that it has diverse therapeutic activity , including anti - inflammatory , antioxidant , antithrombotic , anticancer , cardioprotective , and neuroprotective effects . when used on the skin , fa shows a photoprotective effect [ 2 , 711 ] . in spite of the remarkable pharmacological potential , fa has a poor solubility in aqueous medium   and presents an unfavorable pharmacokinetics which reduces its bioavailability and clinical efficacy after oral administration . in order to increase fa bioavailability and enhance its cytoprotective effects , new formulations have been prepared in which this phenolic acid is entrapped either in solid lipid nanoparticles or in niosomes or is bound to other therapeutic agents through organic moieties ( e.g. , amino acids ) which serve as carriers . however , no paper was devoted to investigate the use of polymeric microparticles as a pharmaceutical approach for improving the therapeutic efficacy of fa by prolonging its retention time into the gastrointestinal tract . these multiple - unit pharmaceutical dosage forms are solid and show spherically shaped particles ranging in size from 1 to 1000  m , in which the drug can be either adsorbed at the surface of the polymer or encapsulated within the polymer . the advantages of using such carriers include potential to increase the bioavailability of poorly water - soluble drugs , ability to control the rate and/or the site of drug release , possibility to improve drug stability related to enzymatic , environmental , or chemical / photochemical degradation , elimination of incompatibilities among different drugs , and as a taste - masking approach [ 14 , 15 ] . various methods are readily available for microencapsulation of drugs and one of the most commonly used is emulsion / solvent evaporation . this procedure can be performed via various protocols and the selection for best option depends on the property of the compounds that are intended to be encapsulated . in our laboratory , experiments are being carried out in order to formulate fa - loaded polymeric microparticles using the simple emulsion / solvent evaporation technique and two biocompatible and biodegradable polyesters , poly(-caprolactone ) ( pcl ) and poly(3-hidroxybutirate - co-3-hidroxyvalerate ) ( phbv ) . however , an analytical validation procedure is required for assaying fa from these formulations at the same time . the literature reports few hplc methods for determining fa from different sample types as summarized in table 1 . these papers are mainly devoted to describing hplc methods for isolation and analytical and bioanalytical quantification of fa from medicinal herbs and foods . only two papers used hplc method for quantifying fa from inclusion complexes that were prepared using cyclodextrins [ 2 , 18 ] . however , none of these two studies involving cyclodextrins were submitted to the required validation process . in that sense , no work was found on validation of analytical methods for determining fa as bulk drug and from pharmaceutical formulations . according to the international conference on harmonization ( ich ) stability guideline , stress testing of a drug substance can be carried out to elucidate its inherent stability characteristics under hydrolytic , oxidative , and photolytic conditions . considering fa , to the best of our knowledge , there is no stability - indicating method previously published for this drug . taking all the above into account , in the current paper , a fast , simple , and optimized reverse - phase hplc method with uv detection was developed and validated for quantifying fa in polymeric microparticles . other experiments were also performed to explore forced degradation of the drug under stress conditions as per the ich guidelines [ 1921 ] . ferulic acid 99.80% ( fa , suzhou leader chemical co. ltda . , suzhou , china ) , poly(-caprolactone ) ( pcl ) ( mw- = 7000090000 louis , mo , usa ) , poly(3-hidroxybutirate - co-3-hidroxyvalerate ) ( phbv ) ( mw- = 380000  g / mol , 8.70  mol% hydroxyvalerate , biocycle l110 , phb industrial , serrana , brazil ) , and poly(vinyl alcohol ) ( pva ) ( mw- = 72000  g / mol , 88.5  mol% of hydrolysis , vetec , rio de janeiro , brazil ) were used as received . as an internal standard , trans - ferulic acid , matrix substance 99.0% ( hplc ) , was purchased from sigma - aldrich ( st . louis , mo , usa ) . hplc - grade methanol was also provided by sigma - aldrich ( st . louis , mo , usa ) . water was purified in a milli - q plus water purification system ( millipore , bedford , ma , usa ) . a varian pro - star sys - lc-240-e hplc system ( walnut creek , ca , usa ) was used for method development . the hplc system was equipped with a column oven compartment ( model prostar 410 ) , an on - line degasser ( model prostar 230 ) , a ternary pump ( model prostar 230 ) , a solvent delivery module ( model prostar 230 ) , an auto sampler ( model prostar 410 ) , and a photodiode array detector ( dad ) ( model prostar 335 ) . data acquisition , analysis , and reporting were performed using varian star workstation chromatography software ( walnut creek , ca , usa ) . experiments were performed in the previously described hplc system using a varian c18 analytical column ( walnut creek , ca , usa ) with 5  m particle size , 4.6  mm internal diameter , and 250  mm length at 25  2c . the mobile phase consisted of methanol and water adjusted to ph 3.0 with orthophosphoric acid 0.1  n ( 48  :  52 v / v ) at an isocratic flow rate of 1.0  ml / min . the method run time was 8 minutes and all experiments were carried out in triplicate . three different formulations ( table 2 ) were obtained for each polymer ( phbv / pcl ) depending on the amount of fa into their compositions ( 5 , 10 , and 20% ) . chloroform and methylene chloride were used as polymer solvent for phbv and pcl , respectively . briefly , the organic phase was added into the aqueous phase under mechanical stirring ( 5000  rev / min ) for 5  min . the emulsion was kept under mechanical stirring ( 800  rev / min ) at room temperature ( 25  2c ) for 4  h. after organic solvent evaporation , microparticles were separated by centrifugation ( 2500  rev / min , 10  min ) , washed twice with purified water , and dried under vacuum at 35  2c for 4  h. the samples were stored into a desiccator under vacuum at room temperature ( 25  2c ) . all procedures were performed in dark conditions . a stock standard solution ( 1  mg / ml ) was daily prepared by dissolving 50  mg of fa into a 50  ml volumetric flask using methanol . this solution was further diluted in methanol to prepare seven different working standard solutions ranging from 10.0 to 70.0  g / ml . these solutions were filtered through a poly(vinylidene fluoride ) membrane filter ( durapore membrane , 0.45  m pore size , millipore , bedford , ma , usa ) before injection into the hplc system . for fa quantification , the amount of drug into fa - loaded phbv / pcl microparticles was indirectly determined . as previously described , microparticles were centrifuged at 2500  rev / min for 10 minutes and washed twice with purified water after organic solvent evaporation . free fa was assayed in the supernatant after its suitable dilution and filtration through a 0.45  m poly(vinylidene fluoride ) membrane filter by injection into the hplc system . the chromatographic conditions were optimized for resolution of the peak of fa by varying the composition and proportion of the mobile phase . the method was then validated and used for the determination of fa into phbv / pcl microparticles . validation studies were performed using the optimized chromatographic conditions based on the principles of validation described in the international conference harmonization ( ich ) guidelines . the method was validated for specificity , linearity , limit of detection ( lod ) , limit of quantitation ( loq ) , accuracy , precision , and robustness . the specificity was determined by analyzing the chromatograms of unloaded microparticles ( m1fa0 and m2fa0 ) in comparison with those obtained for fa - loaded microparticles ( m1fa10 and m2fa10 ) aiming at confirming that none of the excipients interfere with the quantitation of the drug . the linearity was determined by calculating a regression line from the plot of the peak area versus concentration of the working standard solutions prepared at seven concentration levels ( 10.0 , 20.0 , 30.0 , 40.0 , 50.0 , 60.0 , and 70.0  g / ml ) using least - squares linear regression analysis . the solutions were injected in triplicate into the hplc column keeping the injection volume constant ( 10  l ) and chromatograms were recorded . the standard deviation ( sd ) value for the slope and y - intercept of the calibration curve were calculated . lod and loq were calculated based on the standard deviation of the response (  ) and the slope ( s ) of the calibration curve and were expressed as 3.3 /s and 10 /s for lod and loq , respectively . the accuracy of the analytical method was investigated by spiking unloaded microparticles ( m1fa0 and m2fa0 ) with known concentrations of the stock solution to achieve final theoretical drug concentrations of 15 , 45 , and 65  g / ml . the accuracy value was determined by calculating the percent recovery of fa for these three concentration levels and then determining the relative standard deviation ( rsd ) . the precision was assessed at two levels : repeatability ( intraday precision ) and intermediate precision ( interday precision ) using m1fa10 . the repeatability was investigated by testing three different sample solutions at 15 , 40 , and 65  g / ml on the same day . three samples solutions at 40  g / ml were also evaluated in other two different days in order to determine intermediate precision . results were reported in terms of rsd . in order to determine the robustness , experimental conditions robustness was evaluated by analyzing drug content of the microparticles ( m1fa10 ) with variations in the temperature of analytical column ( 30 and 40c ) , flow rate ( 0.9 and 1.1  ml / min ) , and ph of mobile phase ( 4.0 and 6.0 ) . were also carried out in order to provide some information about drug stability and specificity of the proposed method . the standard solution of fa was subject to accelerated degradation by acid , basic , and photolytic conditions . to investigate the acid degradation , 6.25  ml of the stock standard solution ( 1.0  mg / ml ) was diluted into a 25  ml volumetric flask with 0.1  m hcl . this solution was maintained at room temperature ( 25  2c ) and protected from light for 1  h. after the reaction time , the solution was neutralized using 0.1  m naoh . the solution was diluted with methanol to achieve a final concentration of 40  g / ml before injection into the hplc system . the same procedure was used for the alkaline degradation , except by the fact that 0.1  m hcl was replaced by 0.1  m naoh in sample preparation . for photodegradation , 3  ml of fa methanol solution ( 1  mg / ml ) was placed in a quartz cuvette and subsequently exposed to uv radiation ( phillips tuv lamp254  nm , 30  w ) for 6  h in a mirrored chamber ( 1  m  25  cm  25  cm ) at a fixed distance . at predetermined times ( 0 , 1 , 2 , 3 , 4 , 5 , and 6  h ) of exposure to light , 400  l of the samples were withdrawn and diluted with methanol ( final concentration of 40  g / ml of fa ) in order to quantify the remaining fa according to the method previously described . in order to refute the hypothesis of thermal degradation , a cuvette containing fa methanol solution ( final concentration of 40  g / ml of fa ) was covered by aluminum paper and was evaluated as the same way . the degradation rate kinetics of fa was determined and the best fit was used for indicating the reaction order . the kinetic models used were zero order ( c = co  kt ) , first order ( ln c = ln co  kt ) , and second order equation ( 1/c = 1/co + kt ) . in order to demonstrate the applicability of the validated method , the encapsulation efficiency ( ee ) of fa into phbv / pcl microparticles was calculated using ( 1 ) from the hplc results provided by sample solutions:(1)ee%=total  drug  contentfree  drug  contenttotal  drug  content100 . previous tests were carried out in order to provide a quick and effective method for analyzing fa using hplc . initial runs were performed using a mobile phase mixture of acetonitrile  :  water ( 16  :  84 v / v ) containing 1% glacial acetic acid based on a previously reported method for fa quantification in plasma . however , irregular peaks were observed , showing low chromatographic resolution probably due to instrumental and column differences . in order to improve the quality of chromatographic method , various ratios in isocratic mode were tested using different mixtures of acetonitrile  :  methanol  :  water adjusted to ph 3.0 with acetic acid . these assays demonstrated that the proportion of acetonitrile  :  methanol  :  water adjusted to ph 3.0 with acetic acid ( 24  :  24  :  52 v / v ) was more appropriate for the method optimization . in spite of the symmetry of fa peak , therefore , additional experiments were carried out removing acetonitrile and using acetic acid and orthophosphoric acid as acidifying agents at different ph values . the mobile phase composed by methanol  :  water adjusted to ph 3.0 using orthophosphoric acid ( 48 :  52 v / v ) provided a lower tailing and a more symmetric peak for fa with a lower retention time ( theoretical plates = 1253 , k = 3.77 , and tail factor = 1.3 ) . the peak was detected at 4.86  min ( figure 2 ) which is very suitable for routine analyses . in summary , methanol  :  water adjusted to ph 3.0 using orthophosphoric acid ( 48  :  52 v / v ) , column temperature at 25  2c , sample injection volume of 10  l , isocratic flow rate of 1.0  ml / min , detector set at 320  nm , and run time of 8 minutes were chosen as suitable chromatographic conditions for further procedures including the method validation . the proposed method was validated by determining its performance characteristics regarding specificity , linearity , limit of detection , limit of quantification , accuracy , precision , and robustness . specificity was demonstrated by comparing the chromatograms of unloaded and fa - loaded microparticles prepared as per test method . the results showed that there was no interference at the retention time of fa from the other formulation components . in that sense , it is possible to confirm the specificity of the purposed method ( figure 3 ) . moreover , the photodiode array detector indicated that fa peak was free from interference ( purity index > 0.9999 ) . a linear relationship between peak area and concentration of fa at the concentration range of 10.0 to 70.0  g / ml was observed ( figure 4 ) . the linear equation obtained by the least - square method was y = 51.1011x + 26.2381 , where y is the peak area and x is the standard solution concentration in g / ml . a suitable correlation coefficient ( r = 0.9998 ) was recorded which demonstrates that the method is remarkable linear with an r value of nearly 1 at the purposed range . the lowest concentration where fa can be detected ( lod ) and quantified ( loq ) with acceptable precision and accuracy was 0.334 and 1.012  g / ml , respectively . these results represent that the chromatographic method is suitable enough to detect and quantify fa at the concentration range of 10.0 to 70.0  g / ml . the accuracy was evaluated using a recovery study and showed mean recoveries for the three levels of concentration ranging between 99 and 101% ( table 3 ) . these recovery values indicate that the developed method was accurate for the determination of fa in polymeric microparticles . precision was verified by repeatability and intermediate precision as presented in table 4 . the relative standard deviation ( rsd ) the evaluation of robustness was based on rsd values obtained by changing analytical parameters such as temperature of analytical column ( 30 and 40c ) , isocratic flow rate ( 0.9 and 1.1  ml / min ) , and ph of mobile phase ( 4.0 and 6.0 ) . concerning these parameters , the method was considered robust because rsd for the drug content analyses values were lower than 2.24% as summarized in table 5 . therefore , changes in these chromatographic parameters did not affect the analysis of fa into polymeric microparticles . as expected , some variation in the retention time was observed without compromising the determination of drug content . regarding the presence of degradation products , forced degradation study showed different results depending on the stress condition used . after exposure to acid medium , approximately 29.37% of fa was degraded ( figure 5(a ) ) and the drug peak was recorded at 4.56  min . in alkaline condition , a fa degradation of 16.33% was observed and the drug peak was detected at 4.55  min ( figure 5(b ) ) . no previous report was devoted to investigate the drug degradation using 0.1  m hcl and 0.1  m naoh . under photolytic conditions , decreases in fa concentration from methanol solution were observed ( figure 6 ) . on the other side , the cuvette covered by aluminum paper showed no degradation after 6  h which indicates that no thermal degradation was involved in the photodegradation process . moreover , no additional peak was verified in the chromatograms of the forced degradation study demonstrating that the degradation products were not detected using the optimized chromatographic conditions . aiming at elucidating the kinetics of fa photodegradation from methanol solution , the experimental data were fitted to zero , first , and second order equations . these plots ( figure 6 ) indicated that fa photodegradation process in methanol solution followed a second order kinetic with a rate constant of 6.27  10  l / mol  h. in this case , the degradation of fa is dependent on the drug concentration . the drug content and encapsulation efficiency ( ee ) of fa into phbv / pcl microparticles was carried out by the previously validated hplc - dad method and the obtained results are represented in table 6 . high percentages of drug entrapment were obtained for phbv / pcl microparticles by simple emulsion / solvent evaporation . these values are mainly based on the poor aqueous solubility ( 6.63  mg / dl at ph = 7.2 ) of fa   which leads to increase the drug loaded into polymeric microparticles . stearic acid- and stearyl ferulate - based solid lipid nanoparticles containing trans - fa revealed a drug entrapment of 95.4 and 97.7% , respectively . poly(lactic - co - glycolic acid ) nanoparticles containing fa showed ee of about 76% . a fa entrapment higher than 60% was achieved for inclusion complex of trans - fa and hydroxypropyl--cyclodextrin that was prepared by the freeze - drying method . therefore , the validated method was successfully applied to the determination of fa into polymeric microparticles and can be considered an important tool for the quality control of these promising formulations . a simple and efficient reverse - phase hplc - dad method was developed and validated for quantitative determination of fa into polymeric microparticles . in summary , the method was found to be specific , linear , accurate , precise , and robust for a rapid determination of this drug and can be used for studying the stability and degradation kinetics of fa .
a simple stability - indicating hplc - dad method was validated for the determination of ferulic acid ( fa ) in polymeric microparticles . 
 chromatographic conditions consisted of a rp c18 column ( 250  mm  4.60  mm , 5  m , 110   ) using a mixture of methanol and water ph 3.0 ( 48  :  52  v / v ) as mobile phase at a flow rate of 1.0  ml / min with uv detection at 320  nm . 
 the developed method was validated as per ich guidelines with respect to specificity , linearity , limit of quantification , limit of detection , accuracy , precision , and robustness provided suitable results regarding all parameters investigated . 
 the calibration curve was linear in the concentration range of 10.070.0  g / ml with a correlation coefficient > 0.999 . 
 precision ( intraday and interday ) was demonstrated by a relative standard deviation lower than 2.0% . 
 accuracy was assessed by the recovery test of fa from polymeric microparticles ( 99.02% to 100.73% ) . 
 specificity showed no interference from the components of polymeric microparticles or from the degradation products derived from acidic , basic , and photolytic conditions . in conclusion , the method is suitable to be applied to assay fa as bulk drug and into polymeric microparticles and can be used for studying its stability and degradation kinetics .