Focused microwaves-assisted extraction of theobromine and caffeine from cacao

Food Chemistry 129 (2011) 1819–1824

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Analytical Methods

Focused microwaves-assisted extraction of theobromine and caffeine from cacao Leslie N. González-Nuñez, María P. Cañizares-Macías ⇑ Facultad de Química, Departamento de Química Analítica, Universidad Nacional Autónoma de México, México, DF 04510, Mexico

a r t i c l e

i n f o

Article history: Received 20 August 2010 Received in revised form 11 May 2011 Accepted 24 May 2011 Available online 30 May 2011 Keywords: Focused microwaves Extraction Cacao Theobromine Caffeine

a b s t r a c t With the aim of improving the extraction efficiency of Theobromine (TB) and Caffeine (CF) from cacao, a focused microwaves assisted method was developed. To optimize the extraction a 23 factorial design was used with optimum values: 5 min of irradiation time, 210 W of power and 100 mL of the extractant (water). To quantify TB, a study about the reagents that must be added to the extracts (Carrez reagents: (Zn(CH3COO)2 and K4Fe(CN)6) and a Pb(CH3COO)2 solution) in order to eliminate interferences (molecular weight high compounds) was carried out. The extraction method was applied to cacao seeds, cacao powder and cacao peels. The focused microwave method was more efficient compared with a standard mechanic stirring method: for TB from 15.0% (in cacao seeds) to 72% (in cacao peels) and from 36% (in cacao peels) to 153% (in cacao seeds) for CF. Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction Cacao is one of the seeds more consume in the world principally as an ingredient of beverages, pastries or candies. In the last years the consume of cacao has been rising because of its healthy benefits due to its high antioxidant capacity (Abbe Maleyki & Isamil, 2010; Pascual, Valls, & Solà, 2009). There also are some studies which have ranged from a reduction in the likelihood of preeclampsia (Triche et al., 2008) to the inhibition of atherosclerotic plaque progression (Vinson et al., 2006). Cacao has also methylxantines, being TB and CF the most important. This importance is because its incidence over the central nervous system and each person reacts different according to the ingested amount (Yamada, Yamada, Okano, Terashima, & Yokogoshi, 2009). On the other hand, during the harvest and the curing procedure all the cacao components suffer some changes, so the determination of TB and CF has been included in the routine analysis to know the quality of the seed and to avoid fakes in derivative cacao products. The more used instrumental technique to determine TB and CF in cacao is HPLC (Hasegawa, Takahashi, Saijo, Ishii, & Nagata, 2009; Ortega et al., 2010; Ptolemy, Tzioumis, Thomke, Rifai, & Kellogg, 2010; Tzanavaras, Zacharis, & Themelis, 2010) besides there are others instrumental methods with suitable precision: electrochemical (Lee & Kim, 2009; Spãtaru, Sarada, Tryk, & Fujishima, 2002), electrophoretic (Li et al., 2009) and spectrophotometric methods (Khanchi et al., 2007; Shufen, Berger, & Hartland, 1990). ⇑ Corresponding author. Tel.: +52 55 56 22 37 88. E-mail addresses: [email protected], [email protected] (M.P. CañizaresMacías). 0308-8146/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2011.05.118

The extraction time is the most important in the sample treatment when we are analyzing cacao. Therefore it is crucial to improve the extraction efficient of its analytes with the aim of knowing the more precise amount of its in cacao and also know what is the influence of the consumption in health and how are the affected in the curing procedure. Due to the high solubility of TB and CF in water the extraction of these compounds has been carried out using this solvent allowing easy extraction procedures (Brunetto et al., 2007; LoCoco, Lanuzza, & Micali Cappellano, 2007). The microwave scale goes from 300 GHz (infrared radiation) to 300 MHz (radio radiation). The waves of microwave are nonionizant radiations that cause the mobility of the molecules due to the migration of ions and the bipolar rotation. When the electric camp decreases the thermic disorder that produce energy as heat is restored. This law makes that the interaction between the analytes in a matrix and the extractant is better and the extraction is more efficient (Eskilsson & Björklun, 2000; Luque-Garcia & Luque de Castro, 2003). There are some examples of focused microwave extraction that improve the extraction of polar and non-polar compounds in different matrixes as the extraction of ketoprofen in pharmaceutical cream formulation (Labbozzetta et al., 2009), the extraction of heterocyclic amines in meat (Martín-Calero, Pino, Ayala, González, & Alfonso, 2009) or in order to clarify the possible hazard of acidic pharmaceuticals (ibuprofen, naproxen, ketoprofen, and diclofenac) in river water and sediments (Varga et al., 2010). Using microwave in solid samples allows more efficient and faster sample treatment, so it is being more used in analysis laboratories. But in food industry has not been used due to the complexity of the samples, anyway there are some papers talking about this (Ballard, Mallikarjunan, Zhou, & O’Keefe, 2010; Elkhori,

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Jocelyn Paré, Belanger, & Pérez, 2007; Hayat et al., 2009; Moret, Purcaro, & Conte, 2010). As a non-destructive energy, can be applied in food under controlled radiation which has allowed to be used, for example to accelerate the oxidation process in olive oil (Cañizares-Macías, García-Mesa, & Luque de Castro, 2004), or to obtain vanilla extracts with all its properties (Longares-Patrón & Cañizares-Macías, 2006; Valdez-Flores & Cañizares-Macías, 2007). In this paper a new method to obtain TB and CF from cacao samples using focused microwave energy at atmospheric pressure is proposed. This method has been compared with a stirring extraction that was optimized through two methodologies already reported. The obtained extracts were treated with different reagents (Carrez reagents, lead acetate and dihydrogen sodium carbonate) to eliminate the compounds with high molecular weight that interfere in the quantification of TB by UV. To determine CF in the extracts a liquid–liquid extraction with chloroform was done.

2. Material and methods 2.1. Reagents and solutions Different reagents and solutions were used to extract TB and CF from the samples. All reagents used were analytical grade. The following solutions were set to evaluate the used reagents in the stirring extraction methods: (a) Carrez I solution: 24 g of Zn(CH3COO)2(Sigma) were weighed and dissolved into 100 mL of a 3% CH3COOH(Merck) solution. (b) Carrez II solution: 15 g of K4Fe(CN)6(Sigma) were dissolved into 100 mL of water. (c) Basic lead acetate solution: it was prepared by mixing 15 g of anhydrous basic lead acetate (CH3COO)2Pb Pb(OH)2 (Baker-Mallinkroft), with 150 mL of water in a 250 mL flask and then heating and stirring until the colour of the solution became white. Later it cooled down until it reached room temperature and the solution was filtered and the density adjusted to 1.22–1.23 mg mL-1 with distilled water. Solutions at 10% HCl and 1 M NaOH and NaHCO3 powder (Baker-Mallinkroft) were also used. Petroleum ether (Baker-Mallinkroft) to eliminate fats from the cacao seeds was used. An aqueous stock solution of TB with 500 lg mL1 TB (Fluka) to set the TB standard solutions was used. Chloroform as solvent was used in the stock solution of CF (Sigma) with 500 lg mL1. 2.2. Instruments A Maxidigest 301 microwaves device of 300 W from Prolaboto extract TB and CF from the samples using focused microwaves was used. For the extraction by stirring, a magnetic stirrer with controlled temperature (CIMAREC Model so46925) was used. An ultrasonic bath (Branson Model 2510R), a centrifuge (HETTICH EBA 20), an oven (Rios Rocha) and a pHmeter (Oakton) were also used in the treatment of the samples. Statgraphics Plus 4.0 software (Statistical Graphics, Rockville, MD) was used for data analysis. 2.3. Samples The used samples were cacao seeds from Comalcalco, Tabasco, Mexico. The cacao peels were obtained from the analysed cacao seeds. Commercial powder was also analysed.

2.3.1. Samples treatment The cacao seeds were grinded without peel in a ceramic mortar until soft powder to be analysed. Three extractions of 5 mL with petroleum ether were done to quantify the percentage of fats in the cacao seeds. Later, fats were separated centrifuged the extracts for 2 min at 2500 rpm. The moisture content in the cacao seeds was determined in accordance with the Mexican norm (NMX-F-268-1976): 5 g of grinded cacao seeds were weighed and dried in an oven at 103 °C 2 °C for 16 h. The cacao peels were grinded in a ceramic mortar until soft powder to be analysed. 2.4. Extraction of TB and CF from cacao seeds To determine TB by each of the instrumental methods (HPLC, electrophoretic, or spectrophotometric) it is necessary to eliminate interferences with high molecular weight as proteins or polyphenols. There are some reagents that can be added with this aim: Carrez reagents, lead acetates, etc. In this paper the addition of these reagents in two stirring extraction methods already reported was evaluated and a third comparative method was established. Once the treatment of the extracts to quantify of TB and CF was set, a new method using focused microwave to increase the extraction efficiency of the cacao analytes was developed. Cacao seeds were used to optimize the methods. 2.4.1. Stirring Method I (SMI) by Shufen & Berger J. (1990) One gram of sample in a 150 mL flask was weighed. Ninety-six millilitre of distilled water were added and weighed. The mixture was heated to 80 °C for 5 min in a constant stirring. Four millilitres of the basic lead acetate solution were added stirring the mixture. The solution was weighed again when reached room temperature, and distilled water was added to compensate the evaporation. The solution was filtered to remove the precipitate avoiding the first millilitres. NaHCO3 in a ratio of 0.1 g for each 10 mL of filtered was added to remove Pb2+ ions of the solution and to get a PbCO3 white precipitate. The solution was filtered several times to get a crystalline one. 2.4.2. Stirring Method II (SMII) defined by Lo Coco et al. (2007) 0.5 g of sample were transferred into a 150 mL conic flask and 80 mL of distilled water at 80 °C were added. This mixture was heating using a water bath at 80 °C for 60 min with reflux. Later, the mixture was transferred into a 100 mL flask, then 5 mL of Carrez I reagent were added and stirred for one minute. Then, 5 mL of Carrez II reagent were added, stirred and filtered. The resultant liquid was diluted up to 100 mL. 2.4.3. Proposed stirring method (PSM) One gram of sample was added to 80 mL of distilled water heated at 80 °C. The mixture was kept at 80 °C and stirring for 5 min. Five millilitres of Carrez I were added stirring for one minute. The mixture rested until room temperature was reached, later NaHCO3 was adding until the precipitation stopped. The mixture was filtered and the resultant was diluted with distilled water to 100 mL. 2.4.4. Proposed focused microwave-assisted extraction (FMAE) 0.5 g of sample were weighed and set into a collector tube for microwave. 90 mL of distilled water and 5 mL of Carrez I reagent were added. Extraction conditions: 210 W of power and 5 min of irradiation time. When the extraction finished the mixture was cooling down until room temperature, then NaHCO3 was added until the precipitation stopped. The mixture was filtered and the resultant was diluted with distilled water to 100 mL.

L.N. González-Nuñez, M.P. Cañizares-Macías / Food Chemistry 129 (2011) 1819–1824

2.5. Separation and quantification of TB and CF from the extracts Once that the extracts were obtained, 50 mL of them were transferred into to separating funnel of 100 mL capacity and 5.5 mL (aprox.) of the solution of 1 M NaOH to keep the pH between 12.5 and 12.7 were added. Four extractions of 5 mL chloroform stirring on minute and resting for 5 min each were done to extract CF. The extracts were mixed and diluted with chloroform up to 25 mL in a volumetric flask. The aqueous phase was transferred into a 100 mL volumetric flask and diluted to 100 mL with distilled water and filtered. Then 10 mL were transferred into another volumetric flask, and 50 mL of distilled water and 0.55 mL of the HCl solution of 10% were added. Finally, the solution was diluted with distilled water to 100 mL. Suitable dilutions of the extracts to quantify TB (aqueous phase) and CF (organic phase) from the calibrate curves built for each analyte were done. TB was measured to 275.9 nm and CF to 272.7 nm. The detection limit, the slope precision and intercept precision were calculated for each curve. 2.6. Validation of the proposed FMAE method A factorial 23 design with three central points was used to optimize the FMAE method. Power, irradiation time and extractant volume were evaluated. An ANOVA study, parete chart and the equations describing the dependency of the factors on the response variable were calculated using the Statgraphics software. With the objective of evaluating possible changes in the studied analytes by the microwave radiation, two mixtures of CF and TB (10 mg L1 and 25 mg L1) under the same extraction conditions of the samples were analysed. A study of recoveries adding 2.5 mg of each analyte to one gram of cacao seeds was also carried out. To study the intra laboratory reproducibility and repeatability two independence extractions during seven days were also carried out. The results were analysed using an ANOVA test. 2.7. Evaluating the extraction methods A recoveries study analyzing a mixture of CF and TB with 15 mg L1 to evaluate the extraction methods by stirring (SMI, SMII and PSM) was carried out. 5 mg and 12.5 mg of each analyte were added to one gram of the sample cacao seeds to evaluate the effect of the matrix. It was analysed using only the PSM. t tests and F tests to compare the PSM with the reported methods (SMI and SMII) and the FMAE with PSM were done. The extractions and measurements were done in triplicate.

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used to eliminate high molecular weight compounds from the extracts that make interference in the determination were: with SMI, lead acetate and with SMI the Carrez I reagent (Zn(CH3COO)2). As it is necessary to eliminate the added metals to the extracts, complexion reagents as NaHCO3 (for SMI) and K4Fe(CN)6 (for SMII) were used. The lead acetate and theK4Fe(CN)6 (Carrez II) are highly toxic, so the option of changing for another less dangerous was studied. So in the PSM regents from both methods were mixed: Carrez I reagent instead of lead acetate and NaHCO3 instead of K4Fe(CN)6. Unless with the pH that we are working (5.5 pH of the extracts) the solubility constant increases from 1010 (thermodynamic constant) to 105.6 (apparent constant), this value keeps as low as for a stable precipitate be formed between Zn2+ and HCO 3 ions, so it is possible to eliminate the Zn2+ form the extracts without adding K4Fe(CN)6. pH of the extracts was fixed to 12.5–12.7 to assure that TB is stayed in the aqueous phase and CF passed to the chloroformic phase (Shufen, Berger, & Hartland, 1990). In Fig. 1 the results of the found concentration of TB and CF in cacao seeds when SMI, SMII and PSM are used, are shown. The results show that with PSM the obtained concentration are higher than with the other two methods; in that figure are also shown the results when Carrez I and NaHCO3 are not added to the extracts, then, the reported values – caused by the non-eliminate interferences – is almost a 80% higher. For this motive is a must to add these reagents to determine the real amount of TB and CF in the extracts. The precision of each method was between 0.2 and 0.5 mg TB g1 cacao bean and between 0.01 and 0.04 mg CF g1 cacao bean. To assure than the difference between the media of the found values for each of the methods was statistically different the t test was calculated. Firstable, the F test showed that there was no difference in the variances (the calculated F values were lower than the critical value (F2,2 = 39) for each compared method), so t test was calculated for equal variances. The calculated t values were higher than the critical value (t4 = 2.78) in all cases, therefore the results showed that there was statistic difference between the PSM media and the other two methods, so the extraction efficiency of the PSM is better than the SMI and SMII: TB: 9.40% and 17.30%, CF: 25.60% and 39.40%. The analysis result of the 15 mg L1 TB and CF standard showed that when the SMI and the PSM are used, the recoveries are higher than 98% but when the SMII is used the found recoveries are lower

3. Results and discussion 3.1. Calibrations graphics The lineal equation for TB was: Abs = 0.055(±0.0022)[TB] +0.011(±0.0365) with a lineal range between 3 lg mL1 and 30 lg mL1, a detection limit of 0.11 lg mL1and a regression coefficient of 0.9995. For CF the equation was: Abs = 0.036(±0.0017)[CF]  0.008(±0.031) with a regression coefficient of 0.9998, a detection limit of 0.09 lg mL1 and lineal range from 3 lg mL1 to 30 lg mL1. 3.2. Evaluation of the obtained extracts by stirring The SMI and SMII were carried out according with proposed by Shufen, Berger, and Hartland (1990) and Lo Coco et al. (2007) and the PSM was carried out basing on those. The reagents that were

Fig. 1. Concentration of theobromine (TB) and caffeine (CF) in cacao beans using the stirring methods. SMI: Stirring Method I; SMII: Stirring Method II; PSM: proposed stirring method; PSM⁄: PSM without Carrez I solution and NaHCO3 (details in point 2.4 of experimental part).

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than 80%. In this method the extraction is carried out during 60 min with reflux. Due to the stability of the compounds some lost can be found. As the PSM showed better results than the other two methods the effect in the matrix was only evaluated by the PSM. The recoveries for 5 mg and 12.5 mg to study the matrix effect were 93% and 98% for TB and 90% and 96% for CF. 3.3. Optimization of the FMAE method Top and bottom values used in the factorial design were established according to previous studies. 90 W a 210 W were chosen for power. The extractant temperature was controlled near 80 °C because at boiling point there were spattered in the microwave probe causing that part of the sample was not irradiated. So with a power higher than 210 W the boiling point was reached in a few seconds. The minimum value was selected with the aim of increasing the irradiation time in the sample. Water as extractant was selected to extract the analytes. To optimize the extraction medium some tests adding the Carrez I reagent to the extractant before and after the extraction were carried out. The result did not show a significant difference between both options, so the reagent was added before to apply the microwave making the procedure a few faster. The extractant minimum value including the Carrez I reagent was 25 mL and the maximum 100 mL, with this way we were assuring that the microwave were applied in the whole sample. The irradiation time was three minutes maximum and one minute minimum. An experimental design to study the simultaneous effect of the three factors in the extraction of the TB and CF from cacao seeds was used: power (P), extractant volume (V) and irradiation time (IT) (Table 1). An ANOVA test of the experimental design was carried out after the experiments. This test and the answer to the parete charts (Fig. 2A (TB) and Fig. 2B (CF)) confirm the influence between the three parameters. By showing that P and V are the most influencing factors in the extraction of both analytes. The IT is the less influence factor in the extraction being more relevant with CF than with TB. The codified equations describing the dependency of the factors on the response variable were:

TB ¼ 15:134  0:0588xP  1:924xIT  0:114xV þ 0:0416xPxV þ 0:001xPxV þ 0:015xITxV

ð1Þ

CF ¼ 0:917  0:001xP þ 0:019xIT  0:007xV  0:00018  PxIT þ 8:55x105 xPxV þ 5:19104 xITxV

ð2Þ

The surface response charts between IT and V and between P and IT (no showed) were developed through the Eqs. (1) and (2). With them the influence of the three factors in the extraction of

Fig. 2. Standardized parete chart obtained after analysis of the extraction of theobromine (TB) (A) and caffeine (CF) (B) (95% confidence interval) applying focused microwaves. The experimental plan is shown in Table 1.

TB and CF from cacao seeds is confirmed. When solving the equations we found that the optimum values were: P = 210 W, V = 100 mL and IT = 3 min. In parete and surface charts is was appreciate that with higher IT the extraction of TB and CF increases more in the CF, so two other tests increasing the IT 5 min and 10 min we done. Between 5 min and 10 min the difference CF and TB found amount did not change from one time to the other but between 3 min and 5 min there was an increase of 13% for CF and 10% for TB. It was decided that 5 min was the optimum IT to extract both analytes. Finally, the optimum extraction parameters by FMAE were: P = 210 W, IT = 5 min and V = 100 mL. The found recoveries to evaluate the matrix effect for the mixture of CF and TB of 10 mg L1 and 25 mg L1 were: for TB 100.8 ± 0.4% and 99.3 ± 0.4% and for CF 98.5 ± 0.7% and 101 ± 0.3%. The repeatability and reproducibility values, expressed as standard deviation, showed a high precision method for TB 0.1% and 0.12%, and for CF 1.06% and 1.15%, respectability.

3.4. Analysis of the samples Table 1 Factorial design employed to study the simultaneous effect of the three variables of the sampling module. PSM: proposed stirring method; FMAE: focused microwaves assisted extraction; TB: theobromine; CF: caffeine. Test

Power (W)

Irradiation time (min)

Volume of extractant (mL)

TB (mg g1)

CF (mg g1)

1 2 3 4 5 6 7 8 9 10 11

150 90 210 90 90 210 150 210 210 150 90

2 3 1 3 1 3 2 1 3 2 1

62.5 100 25 25 25 25 62.5 100 100 62.5 100

9.60 7.50 12.30 11.30 10.70 13.60 9.70 9.40 15.60 10.50 6.90

0.80 0.56 0.83 0.86 0.74 0.81 0.84 0.72 0.90 0.82 0.50

The used cacao seeds for the analysis were premium quality: moisture (4.3 ± 0.2%), pH (4.5 ± 0.3) and fat (24.0 ± 3.0%) in accordance with the specifications in the Mexican norm (NMX-F-2681976). The obtained results by FMAE method were compared with the obtained by PSM and are shown in Table 2. Table 3 refers the recoveries obtained using FMAE method. In the cacao seeds samples the recoveries were similar to the obtained by PSM (point 3.2). The recoveries from cacao peel and commercial cacao powder were higher than from cacao seeds because of the fat. To evaluate the importance of fat in the extraction procedure of TB and CF fats were removed from cacao seeds and analysed using FMAE method. The amount of TB and CF (30.1 ± 0.20 mg g1, 2.1 ± 0.01 mg g1) were similar than in commercial cacao powder (29.77 ± 0.26 mg g1, 1.97 ± 0.02 mg g1) which is fat free; the recoveries improved and were between 97% and 101%, when fats

L.N. González-Nuñez, M.P. Cañizares-Macías / Food Chemistry 129 (2011) 1819–1824 Table 2 Concentration of theobromine (TB) and caffeine (CF) in the different parts of cacao by the proposed stirring method (PSM) and the focused microwaves assisted extraction (FMAE) method. TB (mg g1) PSM

a

CF (mg g1) FMAE

PSM

FMAE

Cacao seeds 1 21.13 ± 0.37 2 20.33 ± 0.21

24.84 ± 0.27 23.04 ± 0.17

0.92 ± 0.016 0.89 ± 0.010

2.28 ± 0.014 2.32 ± 0.015

Cacao powdera 1 22.26 ± 0.27 2 21.82 ± 0.18

29.77 ± 0.26 30.12 ± 0.30

1.37 ± 0.010 1.42 ± 0.012

1.97 ± 0.019 2.03 ± 0.017

Cacao peel 1 7.50 ± 0.12 2 7.41 ± 0.15

13.01 ± 0.15 12.60 ± 0.24

0.71 ± 0.016 0.73 ± 0.023

0.96 ± 0.011 1.00 ± 0.016

commercial cacao powder.

Table 3 Recoveries of theobromine (TB) and caffeine (CF) using the focused microwaves assisted extraction (FMAE) method and extraction efficiency of FMAE with respect to proposed stirring method (PSM). Extraction efficiency (%)a

Recoveries (%)

Cacao seeds Cacao powderb Cacao peel a b

TB

CF

TB

CF

90.78 ± 1.07 99.22 ± 0.80 98.66 ± 0.11

92.81 ± 0.62 106.63 ± 0.07 104.75 ± 1.19

15.5 35.8 72.0

153.0 43.4 36.1

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eliminate interferences from the extracts due to lead acetate and NaHCO3were used instead of Zn(CH3COO)2 and K4Fe(CN)6. On the other hand, the FMAE proposed method was able to extract a higher amount of these analytes in different sample of cacao than by PSM, demonstrating the microwave improves the extraction causing no modifications in the extracts. For CF the extraction efficiency improved up to 150% (cacao seeds) and for TB up to 72% (cacao peel). When microwave were used the method was being optimized to extract the higher possible amount of TB; once this was reached the method was being improving to extract the higher possible of CF and avoiding to affect the extraction of TB, showing an efficient, precise, fast and easy method. TB and CF are the main alkaloids of cacao. During fermentation and cured procedures these compounds can have modifications so it is important to know their concentrations to establish the cacao quality. The obtained TB and CF concentrations in the cacao seeds were between 1.5% and 3% and between 0.1% and 0.2%, respectability, which shows that the analysed cacao is good quality.

Acknowledgements The authors are grateful for the financial assistance provided by the Dirección General de Asuntosdel Personal Académico (DGAPA) of the Universidad NacionalAutónoma de México (Proyect No. 209707).

With respect to the PSM. Commercial cacao powder.

References were removed from cacao seeds, the extraction procedure was easier, so the errors decreased. As TB and CF are not soluble in petroleum ether we can keep them during the degreasing process.

3.5. Comparative between the PSM and the FMAE method The F and t tests were calculated using values of Table 2. The F test showed that there were no difference in the variances for none of the samples so the t test was calculated basing on equal variance. The media of TB and CF in the samples was the same for both methods as null hypothesis. The results show that the values of the media were statistically different so the FMAE proposed method extracts a higher amount from the analytes than the PSM (the calculated t value was higher than the critical value (t4 = 2.78). In Table 3 the values that indicate the percentage in which FMAE is more efficient than PSM are also shown. These values were calculated using the average of all measurements for each part of cacao. Table 2 shows that the extraction of CF in cacao seeds and cacao powder was more efficient than TB, but in cacao peel the extraction of TB was a little better. The TB/CF ratio by the FMAE method was between 10 and 15 meanwhile by PSM was between 10 and 23, which means a high improving in the extraction of CF by microwave method. On the other hand, these results show the importance of the analysis of these two analytes to avoid frauds in cacao derivates (beverages, pastries, commercial cacao powder, chocolate bars, etc.) in which cacao peel can be used instead of cacao seeds or cacao powder.

4. Conclusions The PSM showed a better efficiency in the extraction of TB and CF from cacao seeds with respect to the methodologies developed by others, moreover decreasing the use of toxic reagent to

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