Food Anal. Methods DOI 10.1007/s12161-015-0207-6
Diazo Coupling Reaction of Catechins and Alkylresorcinols with Diazotized Sulfanilic Acid for Quantitative Purposes in Edible Sources: Method Development and Validation Freddy A. Bernal 1 & Luisa L. Orduz-Diaz 1 & Camilo Guerrero-Perilla 1 & Ericsson D. Coy-Barrera 1
Received: 22 February 2015 / Accepted: 19 May 2015 # Springer Science+Business Media New York 2015
Abstract Catechins and alkylresorcinols are important secondary metabolites distributed in food products, sometimes exploited in quality control and/or as markers. The current methods for their quantitative analysis have some disadvantages such as cost, time consuming, and even insensitive or non-selective. Therefore, suitable, efficient methods are still required. As part of our research on new and rapid methods of analyses in plant and food products, a spectrophotometric method to quantify alkylresorcinols and catechins in plant products was developed and fully validated. The colored product by the diazo coupling between olivetol and catechin with diazotized sulfanilic acid was employed to develop the method. The effect of acid and diazonium salt concentration and the reaction time was analyzed. The method was linear in 0.8–8.3 and 0.6–10.2 μg/mL ranges to olivetol and catechin, respectively. Limit of detection and limit of quantification for olivetol and catechin were 0.253/0.768 and 0.106/0.321 μg/ mL, respectively. Precision at intra-day and inter-day levels (relative standard deviation (RSD) 1.3–9.7 %) and accuracy (99.0–104.9 %) were also demonstrated. Additionally, the application of the method to plant samples containing alkylresorcinol or catechins was subsequently evaluated, being comparable with conventionally employed methods. Thus, the method demonstrated to be fast, easy, inexpensive, and reliable for quantifying these kinds of metabolites in food products.
* Ericsson D. Coy-Barrera
[email protected] 1
Laboratorio de Química Bioorgánica, Departamento de Química, Facultad de Ciencias Básicas y Aplicadas, Universidad Militar Nueva Granada, Cajicá, Cundinamarca, Colombia
Keywords Catechin . Alkylresorcinols . Sulfanilic acid . Diazo coupling . Spectrophotometric method . Validation
Introduction Foods and plant-derived products are commercialized around the world under different policies. Consequently, quality control protocols are not only focused on a single objective. Thereof, the use of appropriate analytical methods is mandatory. There are a great number of protocols intended to determine the type and the proportion of some particular metabolites (chemical markers). Nevertheless, the current quantitative methods can be renewed and improved in order to accomplish a faster and an easier but anyway reliable determination of specific metabolites. Catechins and alkylresorcinols are two kinds of natural compounds particularly important in food-derived products. Catechins are a group of phenolic compounds belonging to the flavonoid family (specifically flavan-3-ol subgroup) which are present in high concentrations in several sources as fruits, vegetables, and beverages. Although catechins are not essential in human nutrition, they help for improving human health on preventing various diseases (Gadkari and Balaraman 2014). These compounds have been extensively studied due to their significant biological potential that includes antioxidant, antiviral, antiplatelet, anticarcinogenic, cardiovascular protective, hypotensive, hypocholesterogenic, and blood sugar level regulator. Moreover, catechin-rich beverages have shown to be effective in reducing body fat (Ogura et al. 2008). Alkylresorcinols are a subclass of phenolic lipids which belongs to the non-isoprenoid category. Some authors have considered alkylresorcinols as fatty acids with a carboxyl group replaced by a 1,3-dihydroxybenzene ring. This feature gives them an amphiphilic nature. Alkylresorcinols are natural
Food Anal. Methods
compounds found in about 11 higher plant families, 5 lower plant families, and several bacterial strains. In animals, there is only one known report (Kozubek and Tyman 1999). On their part, these compounds have been used as biomarkers in the study of the relationship between consumption of whole grain cereals and a decreased incidence of several diseases (Ross et al. 2004). Catechins and alkylresorcinols are frequently analyzed by means of spectrophotometric methods. For catechins, reaction with vanillin/hydrochloric acid and subsequent absorbance measurement at 505 nm has been reported (He et al. 2009). Derivatization with sulfanilamide diazonium salt following absorbance measurement at 425 nm has also been described (Singh et al. 1999). However, these protocols have problems on sensitivity, safety, reliability, and reproducibility. For alkylresorcinols, diazotization with Fast Blue (B and RR) salts and later absorbance measurement at 480/520 nm has been the main method for the last years (Sampietro et al. 2009, 2013). Although this method has important advantages, such as reproducibility and reasonable sensitivity, it could be considered expensive because of the cost of Fast Blue diazonium salts. However, methods based on diazo coupling reactions have significant benefits because they often are faster and involve fewer manipulation steps, the color of the product is more specific and sensitive, and the reagent blank is thus minimized in comparison with other protocols (Gajda et al. 2008; Sampietro et al. 2013). From these facts, HPLC analysis has remained as the preferred technique for the analysis of catechins (Zuo et al. 2002; Mizukami et al. 2007; He et al. 2009; El-Shahawi et al. 2012), and an alternative GC-MS methodology has been proposed to measure alkylresorcinols in the human plasma (Linko et al. 2002). Reported spectrophotometric methods for catechins have also drawbacks such as low selectivity and/or time consuming, while chromatographic methods require several certified standards. In the case of alkylresorcinols, the current method needs specific reaction conditions since the precipitation of insoluble substances can be experienced. Routine methods for quantitative purposes in quality control of catechins and alkylresorcinols in food and plantderived products are still required. Therefore, the aim of this study was to validate a new versatile spectrophotometric method for quantitative determination of catechins or alkylresorcinols in plant extracts based on diazo coupling reaction. The proposed method resulted to be fast, simple, accurate, and precise.
using polymethyl methacrylate (PMMA) and polystyrene (PS) cuvettes. Olivetol and catechin hydrate standards were purchased from Sigma-Aldrich Chemical Co. Ethanol, hydrochloric acid, sulfanilic acid, and sodium nitrite were acquired from Merck Chemical Co. in analytical grade. Preparation of Sulfanilic Acid Diazonium Salt Sulfanilic acid (86.5 mg) was dissolved in hot distilled water and 0.7 % sodium carbonate solution (200 μL). After cooling, the solution was placed into an ice water bath for 10 min, and sodium nitrite (35.0 mg) and 36 % hydrochloric acid (100 μL) were then added. The pale yellow solution was finally quantitatively transferred into a 10.00-mL volumetric flask and made up to the mark with cold distilled water. The final solution was stored at 4 °C protected from light until its usage. Sulfinic acid diazonium salt (SADS) was stable during 2 days under the described conditions. Determination of Wavelength of Maximum Absorption Catechin and olivetol (an alkylresorcinol) stock solutions (60 and 40 μg/mL) were prepared by dissolving the standard in water and ethanol, respectively. The whole process was carried out directly into the cuvettes (ICH Guideline 2005). Catechin Freshly prepared SADS (250 μL) was mixed with 2.4 N HCl (1,000 μL), and catechin stock solution (180 μL) and water (500 μL) were then added. A period of 40 min was allowed to react. Absorbance was measured between 300 and 600 nm against a blank prepared using the same system, but the catechin solution was replaced by distilled water. Olivetol Stock solution of olivetol (180 μL) was mixed with SADS (75 μL), 2.4 N HCl (500 μL) in ethanol and finally made up to 965 μL with ethanol. The reaction mixture was allowed to stand for 80 min, and its absorbance was measured between 300 and 600 nm against a blank prepared using the same system, but replacing the olivetol solution by ethanol. Determination of the Optimum Reaction Time In order to establish the optimal reaction time for the formation of the colored complex, absorbance measurements as a function of time at the maximum wavelength were completed under above-described conditions. Reactions were monitored during 100 min in 30-s intervals (Singh et al. 1999).
Materials and Methods Effect of the Concentration of the Diazonium Salt Reagents and Materials The absorbance measurements and absorption spectra were taken in a Merck Spectroquant® Pharo spectrophotometer
Catechin A set of cuvettes were disposed with different volumes of SADS (20 to 400 μL) and a permanent volume of 2.4 N HCl (1,000 μL). Catechin stock solution (120 μL) was
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then added, and the final volume was kept at 1,930 μL with distilled water. Absorbance was measured at 435 nm after 40 min of incubation. All measurements were carried out as triplicates (Singh et al. 1999). Olivetol A set of cuvettes were disposed with olivetol stock (80 μL). Different volumes of SADS were independently added (10 to 200 μL) to each cuvette, followed by 2.4 N HCl (500 μL) and ethanol up to total volume of 965 μL. Absorbance was measured after 80 min of incubation at 424 nm. All measurements were carried out as triplicates.
absorbance values up to 1. The followed procedure was the same as mentioned for determination of maximum wavelength of absorption. All measurements were carried out as triplicates (ICH Guideline 2005).
Limit of Detection and Limit of Quantitation Ten blanks were prepared to both methods by means of the standard procedure described before. Limit of detection (LOD) and limit of quantification (LOQ) were calculated as follows (ICH Guideline 2005):
Effect of the Concentration of HCl The procedure was performed as indicated for the effect of diazonium salt, but catechin (250 μL) or olivetol (240 μL) with a constant amount of SADS (250 and 150 μL, respectively) and different volumes of 2.4 N HCl (200, 400, 600, 800, 1,000, and 1,180 μL) was then added. For all measurements, a constant volume (1,930 μL) was assured. All measurements were carried out as triplicates (Singh et al. 1999). Determination of the Stoichiometric Relationship Method of continuous variations was used to establish the stoichiometry of both complexes (Ravichandran et al. 2014). Catechin A constant volume of 2.4 N HCl (1,000 μL) was added to a set of cuvettes containing different volumes (12 to 125 μL) of previously diluted SADS (1:20 with distilled water). Different volumes (275 to 35 μL) of 0.92 mM catechin solution were then added to each cuvette. Mixtures were kept up to 1,930 μL with water. The 0 to 1 range of molar fractions between catechin and sulfanilic acid was guaranteed. Absorbance was measured at 435 nm after 40 min of incubation. All measurements were carried out as triplicates. Olivetol Different volumes (35 to 275 μL) of 0.74 mM olivetol solution were added to a set of cuvettes containing different volumes (12 to 125 μL) of previously diluted SADS (1:20 with distilled water). 2.4 N HCl (1,000 μL) and ethanol were then added to each cuvette to reach a final volume of 1, 930 μL. The 0 to 1 range of molar fractions between catechin and sulfanilic acid was guaranteed. Absorbance was measured at 424 nm after 80 min of incubation. All measurements were carried out as triplicates.
LOD ¼
3:3 S m
LOQ ¼
10 S m
where S is the standard deviation of the data and m is the slope of the calibration curve.
Accuracy The accuracy of the method was measured by means of the recovery percentage on spiked samples as follows: % recovery ¼
Aspiked sample −Astandard 100 Asample
Catechin SADS (125 μL) and 2.4 N HCl (500 μL) were mixed in cuvettes, and a catechin sample solution (10 μL) was then added. Increasing amounts of catechin stock solution (20, 25, and 30 μL) were also added. The total volume was kept up to 965 μL with water. The procedure was repeated without the sample solution, and the absorbance was measured at 435 nm after 40 min of reaction. All measurements were carried out as triplicates.Olivetol Increasing amounts of olivetol stock solution (40, 50, and 60 μL) were mixed with an olivetol sample solution (10 μL) in cuvettes. DS (75 μL) and 2.4 N HCl (500 μL) were then added. The total volume was kept up to 965 μL with ethanol. The procedure was repeated without the sample solution, and the absorbance was measured at 424 nm after 80 min of reaction. All measurements were carried out as triplicates. Precision
Linearity For the linearity study, a seven-point calibration curve was constructed using different volumes of stock solutions. 0.6– 10 μg/mL range was employed for catechins. 0.8–8.0 μg/mL range was used for olivetol. These ranges let to cover
The system precision was determined by reproducibility assays through different days and operators. Absorbance measurements to three different concentrations of stocks were determined. Precision was expressed as relative standard deviation (RSD) of four replicates (ICH Guideline 2005).
Food Anal. Methods
Application of the Methods to Natural Product Extracts
Results and Discussion
In order to demonstrate the applicability of the new methods, the quantification of catechin and alkylresorcinol derivatives in two common natural sample-derived extracts was carried out.
Diazo coupling reactions bring to the formation of colored substances named azo compounds. The azo compounds formed between diazotized sulfanilic acid and indole in aqueous solution have been early described in a spectrophotometric method (Ahmad et al. 1986). This report was used as a starting point to build the present method.
Preparation of the Extracts Green tea and wheat bran were selected as representative samples due to their well-known high concentration of catechin and alkylresorcinol derivatives, respectively. These products were then purchased in local markets at Bogotá. Green tea was submitted to extraction by maceration with water at room temperature during 3 h. The extract was filtered and diluted to the mark in a volumetric flask. A dilution (1:10) of this solution was used in the analytical procedure. For its part, wheat bran was exhaustively extracted with acetone. The extract was filtered and concentrated under reduced pressure. An exact amount of extract was weighted and subsequently dissolved in methanol and then transferred to a volumetric flask and finally diluted to the mark.
Quantification of Catechins Total catechin content in the green tea extract was determined as follows: SADS (250 μL) and 2.4 N HCl (1, 000 μL) were mixed, and the extract (50 μL) was then added. Total volume was kept up to 1,930 μL with water. The mixture was let to stand during 40 min, and then absorbance was measured at 435 nm. This value was interpolated in the calibration curve and expressed as milligram catechin equivalent per gram of dried sample. Total catechin content was also determined by means of vanillin method according to the literature (He et al. 2009). All measurements were carried out as triplicates.
Determination of Wavelength of Maximum Absorption The diazo coupling reaction between sulfanilic acid diazonium salt and catechin afforded a colored complex with an absorption maximum at 435 nm as shown in Fig. 1. The absorption spectra for the reaction product between sulfanilic acid diazonium salt and olivetol resulted in a more complex spectrum with a maximum value around 424 nm (Fig. 1). Water and ethanol for catechin and olivetol were respectively used for all analyses. These selections were based on the solubility of the standards and the related analytes in the mentioned solvents (catechins are easily extracted with water, meanwhile alkylresorcinols are preferably extracted with low-medium polar organic solvents). Colored products of both compounds have nearby maximums of absorption. Although this fact could mean a problem in simultaneous quantification of catechins and alkylresorcinols, polarity differences between these compound types result in a highly improbable simultaneous extraction of them. Moreover, despite alkylresorcinols have been reported in a great variety of plant families (Kozubek and Tyman 1999), cereals are those with higher concentration of alkylresorcinols (Ross et al. 2003), bringing even to the definition of alkylresorcinols as biomarkers in wholegrain wheat and rye (Ross 2012). For its part, catechins are polar compounds found in completely different plant families, mainly in tea leaves,
Quantification of Alkylresorcinols Total alkylresorcinol content in the wheat bran extract was determined as follows: extract (50 μL) and SADS (150 μL) were mixed together with 2.4 N HCl (1,000 μL). Ethanol was added to keep the total volume in 1,930 μL. The mixture was let to stand during 80 min, and then absorbance was measured at 424 nm. This value was interpolated in the calibration curve and expressed as milligram olivetol equivalent per gram of dried extract. Total alkylresorcinol content was also determined by means of the Fast Blue RR method according to the literature (Sampietro et al. 2009). All measurements were carried out as triplicates.
Fig. 1 Absorption spectra of the reaction product of olivetol (solid line) and catechin (dashed line) with SADS
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red wine, broad beans, black grapes, apricots, and strawberries (Gadkari and Balaraman 2014). Optimization of Reaction Conditions Concentration of acid, concentration of SADS, and reaction time were considered and analyzed in order to establish the optimal reaction conditions to the present analytical purposes. Temperature was ever kept at 4 °C due to the well-known thermal lability of the diazonium compounds with chloride as a counterion (Bonin et al. 2011). Reaction Time The optimal incubation period for the reaction was separately determined to each analyte. The absorbance of the colored product was measured as a function of the time of the reaction (Fig. 2a). Catechin exhibited a faster reaction than olivetol; therefore, 40 min was the optimum time period for the first one, while 80 min was required to the last one. Olivetol reaction product continued showing a smoothed absorbance increasing beyond 80 min probably due to the solvent evaporation. Hydrochloric Acid Concentration The stability of diazonium salt and colored azo product is recognized as pH dependent and, therefore, is really important to evaluate the influence of the concentration of the acid on the absorbance measurements. Different volumes of 2.4 N HCl were used to evaluate the effect of the concentration of the acid on the reaction medium (Fig. 2b). Acid concentration demonstrated to be a very close behavior on the two analyzed systems. Indeed, acid concentration was only defined as 1.2 N to both methods. Diazonium Salt Concentration In order to assure quantitative reaction of the analyte, the effect of the concentration of the SADS on the cuvette was evaluated. Steady state was reached approximately from 150 μL of SADS solution to the olivetol system as shown in Fig. 2c. For its part, 250 μL of SADS solution was sufficient to the catechin system. Absorbance of the colored product was not significantly affected by higher SADS volumes than 250 μL in both cases. Therefore, these volumes were selected as the optimal quantity of SADS, respectively, for performing adequate analytical methods. Stoichiometric Relationship In order to establish the stoichiometry of the reaction, the continuous variation method (Job’s method) at optimum
Fig. 2 Validation of the method for olivetol (solid line) and catechin (dashed line) with SADS. a Kinetics of the reaction. b Effect of the concentration of acid. c Effect of the concentration of diazonium salt
conditions was used (Ravichandran et al. 2014). The results are shown in Fig. 3. Nearly symmetrical bell shape for both compounds let to establish that catechin:SADS and olivetol:SADS ratios were 1:1. The limiting logarithmic method (Rose 1964) was used in order to confirm the stoichiometric relationship exhibited in Job’s plot. Straight lines were found in all cases, and the slope values near to 1 undoubtedly confirmed the 1:1 ratios. Based on these ratios and the knowledge of the diazo coupling reaction, a pathway was proposed as shown in Fig. 4. Although the proposed azo products are resulting from the coupling in ortho-position to the
Food Anal. Methods
Fig. 3 Job’s plot for the colored products of the reaction of olivetol (filled square) and catechin (filled triangle) with SADS
two hydroxyl groups, the product with azo group at position 4 (in olivetol) and position 8 (in catechin) could also be formed. Method Validation Linearity, Limit of Detection, and Limit of Quantitation Evaluation of the linearity of the method was evaluated by means of Ringbom plots (data not shown). The LambertBeer law compliance was thus restricted to a determined range of concentration. Catechin and olivetol demonstrated to be in agreement with Lambert-Beer law in similar concentration range (Fig. 5). High values to the coefficient determination let to confirm linearity to the analyzed systems. From the slope of the calibration curves, LOD and LOQ were established. Calculated LOD and LOQ for catechin resulted to be lower to those of olivetol. In other words, the sensitivity of the method of catechin is higher than that of olivetol. However, the sensitivity of both methods is acceptable for analytical purposes. Statistical parameters of linearity and sensitivity are summarized in Table 1.
Fig. 5 Calibration curve for olivetol (filled square) and catechin (filled triangle). Marks correspond to the mean of triplicates
Precision The precision of the method was evaluated in intra-day and inter-day assays and expressed in terms of RSD. Analyses of three different concentration solutions containing catechin or olivetol were submitted to the quantitation using the proposed method under optimal conditions, and the results are shown in Table 2. Good intra-day precision was established for the two analytes (RSD lesser than 5 %), although particular tendency was not rendered clear. However, inter-day precision for catechin quantification resulted to be better than that of olivetol.
Accuracy The accuracy of the method was determined by means of recovery test. Samples of catechin and olivetol were spiked with standard solutions at three different concentrations and then analyzed using the proposed method. The results are presented in Table 3. The recovery percentages ranged from 99.0 to 103.3 and from 99.5 to 104.9 for catechin and alkylresorcinol quantification methods, respectively. Although these results were
Fig. 4 Proposed reaction pathways between olivetol (upper) and catechin (lower) with SADS
Food Anal. Methods Table 1 methods
Comparative linearity and sensitivity chart for the proposed
Parameter
Total catechins quantitation method
Total alkylresorcinols quantitation method
Standard Reaction time (min) λmax (nm) Linear range (μg/mL) Slopea Intercepta Determination coefficient, r2 LOD (μg/mL) LOQ (μg/mL)
Catechin hydrate 40 435 0.0–10.0 0.107 (0.002) 0.0217 (0.0112) 0.9983
Olivetol 80 424 0.0–5.0 0.121 (0.002) 3.18×10−3 (0.008) 0.9988
0.106 0.321
0.253 0.768
Table 3
Accuracy of the methods by recovery test
Analyte
Sample concentrationa (μg/mL)
Standard added (μg/mL)
Recovery ± RSD (%)b
Catechins
2.6
Alkylresorcinols
3.9
2.5 3.1 3.7 1.6 2.1 2.5
101.3±4.0 100.9±2.9 99.0±2.0 99.5±7.9 102.9±5.4 100.9±3.9
a
Sample of catechin hydrate and olivetol to catechin and alkylresorcinol quantification, respectively
b
Result of three determinations
a
Slope and intercept of the calibration curve built with microgram per milliliter as concentration units. The value is represented as a mean (uncertainty) of triplicates
highly variable, recovery percentages let to establish an acceptable accuracy for the proposed methods. Application of the Methods to Real Samples In order to demonstrate the performance of the present method, real samples were submitted to quantification process. The results were compared with those obtained from conventional spectrophotometric methods (Table 4). Quantitative results (obtained by means of the novel diazotized sulfanilic acid method herein proposed) demonstrated to be reproducible and comparable with those from conventional spectrophotometric methods in spite of completely different action mechanisms. For its part, catechin quantitation was highly closed for both conventional and novel method. At the same time, problems by
Table 2 Intra-day (repeatability) and inter-day (intermediate precision) precision of the methods Analyte
Intra-day precision Exp. conc.a Sb (μg/mL)
Catechin 1.57 3.92 6.14 Olivetol 1.44 3.18 5.29
0.06 0.05 0.13 0.05 0.13 0.07
chemical interferences in real matrices were not exhibited and could be stated as negligible. However, significant differences were evident from Student’s t test between methods, although conventional methods are not necessary absolute neither accurate. Thus, the method is easier than the other protocols because the described procedure requires less manipulation and it is only necessary that the preparation of the reagent solutions be combined to the reaction. In addition, the cost of the reagents is pretty low in comparison with the other methods using expensive reagents. Therefore, the usefulness of these new methods is evident in routine natural product and food chemistry studies. Moreover, the application of the new methods presented here could be extended to metabolic profiling and metabolomics due to their comparable, precise, and rapid results as well as for being cheaper and/or easier protocols.
Table 4 samples
Quantification of catechins and alkylresorcinols in real
Sample
Analyte
Inter-day precision RSD (%) Exp. conc.a Sb (μg/mL)
RSD (%)
4.14 1.30 2.17 3.53 4.20 1.36
3.23 1.66 1.42 5.22 9.72 6.44
1.56 3.91 6.17 1.43 3.45 5.62
0.05 0.06 0.09 0.07 0.33 0.36
Wheat bran extract Green tea leaf extract
Alkylresorcinols Catechins
Measured valuea ± standard deviation Present method
Conventional methodb
15.7±0.8 3.07c ±0.03 6.68d ±0.08
13.6±0.6 3.62c ±0.05 6.93d ±0.21
a
Expressed in milligram olivetol equivalent per gram of dried extract for wheat bran extract and milligram catechin equivalent per gram of dried sample for green tea leaves
b
RSD relative standard deviation
Fast Blue RR method for olivetol and vanillin/sulfuric acid method for catechin
a
Mean of four determinations
c
Water as an extraction solvent
b
Standard deviation
d
Water:ethanol (1:1) as an extraction mixture
Food Anal. Methods
Conclusions Through the present study, a novel, rapid, and easy method for quantification of alkylresorcinols and catechins via diazo coupling reaction has been developed. This spectrophotometric method demonstrated to provide reproducible and accurate results using a simple, sensitive, and rapid procedure. The reagents employed in the proposed methods are cheap and readily available, and the protocol developed involves simple experimental conditions and in situ preparation of the chromogenic reagent. The proposed method also exhibited comparable analytical performances with conventional methods for quantification of alkylresorcinols and catechins and proved to provide adequate analytical results on wheat bran and green tea extracts as representative real samples. Therefore, the proposed method can be used as a routine method in quality control and chemical profiling for natural product extracts and foods.
Acknowledgments The authors thank MU Nueva Granada for financial support. The present work is a product derived by the Project INVCIAS-1471 financed by Vicerrectoría de Investigaciones at UMNG— Validity 2014. Conflict of Interest The authors declare that they have no conflict of interest. This article does not contain any studies with human or animal subjects.
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