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the polyphenols that are present in foods can be divided broadly into two main groups : flavonoids and other compounds that are mostly monocyclic aromatic acids .
cocoa and chocolate products have the highest concentration of flavonoids among commonly consumed foods and also have many health promoting properties . over 10 percent of the weight of cocoa powder
cocoa and chocolate are among the most concentrated sources of the procyanidin flavonoids , catechin , and epicatechin .
for example , dark chocolate has been found to be very nutritious , potent antioxidant , that improves blood flow , lowers blood pressure , raises hdl , protects ldl against oxidation , lowers the risk of cardiovascular disease , protects the skin against the sun , and improves blood flow to the brain .
epicatechin has proved that consumption of plain , dark chocolate resulted in an increase in both the total antioxidant capacity and the (  ) epicatechin content of blood plasma but that these effects are markedly reduced when the chocolate is consumed with milk or if milk is incorporated as milk chocolate . a study conducted over a two - week period in 21 healthy adult subjects with a daily consumption of high - flavonoid ( 213  mg procyanidins , 46  mg epicatechin ) or low - flavonoid dark chocolate bars ( 46  g ,
1.6  oz ) observed improvement in endothelial function associated with an increase in plasma epicatechin concentrations and no changes in oxidative stress measures , lipid profiles , blood pressure , body weight or bmi were observed .
epicatechin has also been proved to improve vascular functions and insulin sensitivity , to reduce blood pressure and platelet reactivity , and to be good antioxidant [ 37 ] .
the growing interest in these compounds and the number of methods for quantification in various herbs , herbal extracts , herbal formulations , and food items have been developed .
a thorough literature search revealed that the high - performance liquid chromatographic ( hplc ) methods are more predominant .
several hplc methods with different detection modes , ultraviolet [ 813 ] , photodiode array [ 14 , 15 ] , electrochemical [ 10 , 16 , 17 ] , capillary electrophoresis [ 18 , 19 ] , fluorescence [ 10 , 20 ] , and mass [ 21 , 22 ] detection , as well as few high - performance thin layer chromatographic ( hptlc ) methods [ 23 , 24 ] , are also reported . in view of the growing interest in epicatechin
, we report a simple , rapid , and precise gradient hplc method with an economical mobile phase for the simultaneous separation and quantification of catechin and epicatechin , in cocoa powder and chocolates .
all hplc grade solvents ( acetonitrile , methanol , water , and orthophosphoric acid ) were purchased from qualigens ( mumbai , india ) .
standard catechin and epicatechins were prepared by natsol laboratories private limited , visakhapatnam , india .
identity and purity ( > 98% ) of the compounds were confirmed by chromatographic ( hplc ) and spectral ( ir , mass , and nmr ) data . the hplc system ( shimadzu , 2010 cht ) consisted of quaternary pump with vacuum degasser , thermostatted column compartment , autosampler , and uv detector .
a reverse - phase column ( targa , c18 , 5   , 250  4.6  mm ) was used and the column temperature was maintained at 30c .
solution a : 0.1  ml of orthophosphoric acid dissolved in 900  ml of hplc grade water and the volume was made up to 1000  ml with water and the solution was filtered through 0.45  m membrane filter and degassed in a sonicator for 3 minutes , solution b : acetonitrile .
mobile phase was run using gradient elution : at the time 0.01 minutes 11% b ; at the time 30 minutes 25% b ; at the time 35 to 39 minutes 100% b ; and at the time 40 to 50 minutes 11% b. the mobile phase flow rate was 1.0  ml / minute and the injection volume was 10  l .
stock solutions of catechin and epicatechin ( 4  mg / ml ) were prepared by dissolving standards into methanol .
50  mg of the sample was transferred into a 100  ml volumetric flask containing 75  ml of methanol , sonicated for 10 minutes , diluted to 100  ml with methanol , and filtered through 0.45  m membrane filter .
about 10 gm of chocolate sample was extracted with methanol on a hot water bath till the colorless methanolic extract was obtained .
calibration standards were prepared by diluting the catechin and epicatechin stock solutions with methanol in the concentration range of 100600  g / ml . the standard curves were obtained by using the peak areas of six different concentrations in six replicate assays and were expressed by the linear least square regression equation .
the peaks for catechin and epicatechin in the samples were confirmed by comparing the retention times of the peak with those of standards .
the linearity of the method was checked with catechin and epicatechin standards with the calibration curves in the range 100600  g / ml , using six different concentrations in six replicate assays .
limit of detection ( lod ) and limit of quantification ( loq ) were obtained from the standard deviation (  ) of the blank response ( n = 6 ) and slope ( s ) of the calibration curve using the formulae 3.3 /s and 10 /s , respectively .
accuracy was determined by spiking preanalyzed sample with low , medium , and high concentrations in the calibration range of the standard catechin and epicatechin and analyzed by the proposed method .
the experiments were conducted six times to check the recovery of the analyte at different levels in the samples .
precision is a measure of repeatability of the analytical method in the normal operating conditions .
precision ( intra- and interday ) of the method was verified by six determinations of three concentrations of the standard catechin and epicatechin on the same day ( intraday ) and on different days ( interday ) which were carried out and expressed as percent relative standard deviation ( % rsd ) .
a simple method was developed for the determination of catechin and epicatechin based on a reversed phase hplc separation combined with ultraviolet detection .
the gradient system was chosen to minimize the variations of the baseline , considering the simplicity , precision , and accuracy .
the stability of the samples and standards of catechin and epicatechin were checked and found to be stable for 12 hours . a systematic study conducted on the effect of column selection and mobile phase composition on the separation of catechin and epicatechin based on the resolution and peak shapes .
best results were obtained with targa c18 column , with 5  m particle size , 250  mm length , and 4.6  mm i.d , and a gradient system of mobile phase comprises 0.1% ( v / v ) .
phosphoric acid in water ( mobile phase a ) and acetonitrile ( mobile phase b ) with 1.0  ml / minute flow rate enable the baseline separation of catechin and epicatechin within 50 minutes .
all the separations were performed at 30c , absorption measurement at 280  nm was selected , and the compounds catechin and epicatechin were effectively detected ( figure 1 ) .
method for quantitative analysis of catechin and epicatechin was validated with regard to its specificity , linearity , accuracy , and precision by utilizing the ich guidelines .
the specificity of the method was confirmed by reliability of compound peaks corresponding to catechin and epicatechin in the sample .
the sample was spiked with standard catechin and epicatechin , individually , which were separated by gradient mobile phase system .
the peak areas of the catechin and epicatechin in the sample were changed consistently with their corresponding standards .
the study confirmed the stable retention times of catechin and epicatechin in the samples which were comparable to retention times of standards with 0.2 to 0.3 minutes of variation ( table 1 ) .
the linearity of the method was checked with standards corresponding to catechin and epicatechin with the calibration curves in the concentration range 100600  g / ml . the regression equation and the correlation coefficient were observed with six replicate analyses for each concentration .
the results of linearity data were reported in ( table 2 ) showing that the correlation coefficient of the equation was > 0.99 and the two calibration curves yielded straight lines in a wide range .
the limit of detection ( lod ) of catechin and epicatechin was 0.012  g / ml and 0.15  g / ml , respectively , and the limit of quantification ( loq ) of the catechin and epicatechin was 0.036  g / ml and 0.45  g / ml , respectively .
accuracy of the analytical method was determined by spiking sample with known amounts of catechin and epicatechin at three concentration levels .
the samples were analyzed under optimized conditions and the recovery rates were observed in the range 99.98% to 100.24% for catechin and 99.48% to 100.26% for epicatechin
the average recovery percentage values were found to be 100.06% and 99.91% for catechin and epicatechin .
the results depicted in ( table 4 ) showed that no significant intra- and interday variations were observed in the analysis of catechin and epicatechin .
the method developed was applied to analyze cocoa powder and ten different chocolates which were procured from domestic market and united states of america .
the results presented in ( table 5 ) reveal that epicatechin is present in higher concentrations than catechin in the cocoa powder and chocolates .
the presence of excipients like sugar , milk fat , and vanilla does not interfere in the determination of catechin and epicatechin in chocolates .
the growing importance of epicatechin as a constituent of therapeutic significance necessitated the development of highly sensitive and selective methods .
a simple reversed phase hplc method has been developed and the results demonstrate that the method developed was highly specific , accurate , and precise and it could be used in the determination of catechin and epicatechin content in chocolates .
interestingly , the branded chocolates contain higher levels of catechin and epicatechin . chocolates of indian origin contain lesser content of catechin and epicatechin comparable to the brands available in usa .
a simple and sensitive reversed phase high - performance liquid chromatographic ( hplc ) method has been developed for the determination of catechin and epicatechin in cocoa powder and chocolates . 
 the separation was achieved on a reversed phase c 18 column ( targa ) 5  m by gradient elution with a flow rate of 1.0  ml / minute with an operating temperature of 30c and detection with a uv - visible detector was at 280  nm . 
 the method was validated for linearity , precision , intra- and interday precision , and accuracy . 
 the developed method is successfully applied for the determination of catechin and epicatechin content in chocolates . 
 the godiva brand chocolate contains high concentration of epicatechin .