An industry consensus study on an HPLC fluorescence method for the determination of (±)-catechin and (±)-epicatechin in cocoa and chocolate products

Background This manuscript describes the results of an HPLC study for the determination of the flavan-3-ol monomers, (±)-catechin and (±)-epicatechin, in cocoa and plain dark and milk chocolate products. The study was performed under the auspices of the National Confectioners Association (NCA) and involved the analysis of a series of samples by laboratories of five member companies using a common method. Methodology The method reported in this paper uses reversed phase HPLC with fluorescence detection to analyze (±)-epicatechin and (±)-catechin extracted with an acidic solvent from defatted cocoa and chocolate. In addition to a variety of cocoa and chocolate products, the sample set included a blind duplicate used to assess method reproducibility. All data were subjected to statistical analysis with outliers eliminated from the data set. Results The percent coefficient of variation (%CV) of the sample set ranged from approximately 7 to 15%. Conclusions Further experimental details are described in the body of the manuscript and the results indicate the method is suitable for the determination of (±)-catechin and (±)-epicatechin in cocoa and chocolate products and represents the first collaborative study of this HPLC method for these compounds in these matrices.


Background
The use of cocoa has been documented for almost 4,000 years. The first population thought to consume the material was the Mesoamericans [1,2]. In the past decade there has been increasing interest and numerous publications on the putative health effects associated with the moderate consumption of cocoa and chocolate products [3][4][5]. In a parallel fashion, several groups initiated studies into the potential agents responsible for cardiovascular effects with the flavanols, (±)-catechin and (±)-epicatechin being candidate compounds [6].
The growing interest in these compounds resulted in a plethora of methods for quantification in various foodstuffs including tea, wine, grapes and chocolate. While other analytical methods have been used, HPLC was the predominant method developed [7][8][9][10][11][12][13]. A thorough literature search using Google and Pubmed resulted in thousands of citations on polyphenol analysis and almost 900 citations on flavanol analysis by HPLC in chocolate, indicating the recent explosive growth in methods for these analytes. Considering the increased interest in the cocoa flavanols' potential cardiovascular effects, a standard quantification method would be pertinent for accurate determination of dose-response effects in clinical trials. There is both an ISO and an Institute for Nutraceutical Advancement (INA) method for flavanols in tea, but not yet a standard method for flavanol quantification in chocolate and cocoa [14,15].
With this as background, the National Confectioners Association (NCA) convened an analytical chemistry working group to develop a consensus HPLC method. This group conducted a collaborative study using samples provided by NCA to establish a method to quantify (±)-catechin and (±)-epicatechin in cocoa and chocolate and make it available to the industry.

Scope and Applicability
This method is applicable for the analysis of (±)-epicatechin and (±)-catechin in cocoa powder, chocolate liquor and formulated chocolate products. This ring trial only included pure chocolate; any products containing inclusions (such as fruit or nuts) may not be appropriate for this method due to potential interference.

A. Principle
This method determines the (±)-catechin and (±)-epicatechin content of cocoa and chocolate products. Fat is removed from the sample in order to prevent potential interference and protect the column by using multiple hexane extractions. Defatted samples are then dried for subsequent extraction of analytes. Defatted, dried samples are extracted, with sonication, at 40°C for 15 minutes using an acetone: water: acetic acid (70: 29.5: 0.5) solvent mixture. Extracted samples are then centrifuged to remove insoluble materials and brought up to a defined volume. The extracts are filtered into HPLC vials for chromatographic analysis. (±)-Catechin and (±)-epicatechin are separated by a reverse phase mechanism on a C18 column with an acidic acetonitrile-water mobile phase gradient. Analytes are detected and quantified by their fluorescence, with excitation at 280 nm and emission at 315 nm.

C. Reagents
(a) Water: High purity deionized water, filtered through a 0.45 μm or smaller pore filter (b) Acetonitrile: HPLC grade (c) Hexane: HPLC grade (d) Acetone: HPLC grade (e) Acetic Acid: Glacial (f) Extraction Solvent: Mix 700 mL Acetone, 295 mL Water and 5 mL Acetic Acid (g) Mobile Phase A: 0.2% Acetic Acid in Water. Add 2 mL Acetic Acid to 1 L Water.
(h) Mobile Phase B: 0.2% Acetic Acid in Acetonitrile. Add 2 mL Acetic Acid to 1 L Acetonitrile. (i) Standards: (±)-Catechin hydrate, purity ≥ 98%, Sigma-Aldrich C1251-5G or equivalent; (±)-epicatechin, purity ≥ 98%, Sigma-Aldrich E4018-1G or equivalent. Certificate of analysis from supplier is required for purity correction of each new lot number. (e) Working standards: Prepare Working Standard Solutions from the chosen stock standard. Add indicated amounts of appropriate working standard (100 or 1000 μg/mL) to a 10 mL volumetric flask; bring to volume with the extraction solvent. Transfers should always be made with Class A volumetric pipettes. Alternatively test tubes can be used and the remaining extraction solvent for dilution to 10 mL can be added with Class A volumetric pipettes. See an example of the working standard dilution scheme in Table 1 below.

D. Standards and Reagent Blank Preparation
(f) Calculate the exact concentration of each component of the working standards as follows: (g) Remove the cap and allow the residual solvent to evaporate in an appropriate fume hood until remaining hexane has evaporated (e.g. over night). Alternately, a stream of nitrogen may be used to accelerate the drying process.

F. Preparation of Test Solutions
Continue with whole sample remaining in the centrifugation tube.
(a) Add 2 glass beads to the centrifuge tube containing the dried, defatted sample.
(b) Add 9 mL of extraction solvent (dispenser) and vigorously shake the sample to break centrifugation pellet. Sample does not need to be completely suspended yet. Shake headlong, if necessary gently tap several times.   Table 2 for example gradient conditions. HPLC columns differ in their selectivity for these compounds. Gradient conditions should be altered as needed to achieve resolution of (±)-catechin and (±)-epicatechin from interfering peaks.

(f) Concentration of analytes in sample extract:
Check if concentration of analytes in sample extract lie within their calibration ranges. If necessary, dilute and re-run extraction solution.
(g) Check sample: Check sample by re-runing mid point calibration curve in middle and at end of sample sequence. Calculate the mean, standard deviation and coefficient of variation (%CV) of the peak areas.

H. Calculations
Integrate peak area for quantitation. If peak areas of analytes in sample extracts are above calibration curve, dilute sample extract solution with extraction solvent accordingly. If peak areas of analytes in sample extracts are below calibration curve, repeat sections E and F and increase sample weight accordingly. Construct standard curves, plotting calibration standard concentration of each standard against the area of the standard peak, using linear regression. Calculate the analytes (±)-catechin and (±)-epicatechin in the original sample as follows:

I. HPLC System and Column Performance Criteria Qualification
An HPLC column which fully resolves the analytes of interest may be used for the method. Gradient slope, flow rates and injection volumes may be altered as appropriate to accommodate columns of differing dimensions.

J. System Suitability
System suitability is a required procedure to ensure the HPLC system is working correctly. The following suitability tests are recommended to ensure correct system operation prior to initial use: Repeatability and carry-over: Before running any test solutions, demonstrate the repeatability and lack of carryover of the HPLC system as follows: (a) System Artifacts: As the first two injections of the day, analyze the blank standard twice in succession. Inspect the two chromatograms for artifact peaks from the HPLC system. Artifacts in the first chromatogram, absent in the second, indicate a buildup of impurities on the system. Artifacts present in both runs indicate impurities expected in every run. If the first chromatogram shows artifact peaks but the second chromatogram does not, inject a blank solution as the first sample in every analytical set. The presence of artifact peaks indicates impurities in the HPLC solvents, the needle wash system, or carryover in the injection system. These problems, if present, should be corrected. (b) Carryover: Inject Standard 5 and then the blank. Carefully examine the blank injection for carryover peaks. Calculate the carryover of any peaks seen in the blanks as a percentage of the concentration found in standard 5. Carryover of standard 5 to the blank injection should be less than 0.1%.
(c) Linearity of the standard curve: Analyze each of the 5 standards, and construct a standard curve. The R 2 of each standard curve should be greater than 0.9990. If this linearity is not achieved, prepare fresh standards.
(d) System precision: Analyze five replicate analyses of standard number 3. Calculate the concentration of each analyte and calculate the mean, standard deviation and percent coefficient of variation (%CV) of the results. The %CV for all peaks should be ideally less than 2%.

K. Samples
Samples for analysis were prepared by the NCA Study Director and submitted as blind samples to five participating laboratories. Samples consisted of cocoa, dark chocolate, milk chocolate and NIST SRM [2384] Baking Chocolate having certified values for (±)-catechin and (±)-epicatechin. One dark chocolate samples was used as a blind duplicate to assess method repeatability.

L. Quantification
Quantification was performed using the external calibration method as described in the method with all laboratories reporting regression coefficients in excess of 0.99 with the labs equally divided whether calibration was forced through zero.

Results and Discussion
All results were submitted to the Study Director using the form that was provided with the samples and with all data statistically evaluated. Samples were run in duplicate or triplicate. Furthermore each data set was evaluated using the Q-test to test for outliers with some data being eliminated. The results can be seen in Table 3. Figure 1 is a sample chromatogram provided from one laboratory of a dark chocolate extract. Instrumental conditions are as described in the methods section.
Each laboratory also provided information about LOD (Limit of Detection) which was in pure solvent in the 40-50 ng/mL. The LOQ (Limit of Quantitation) ranged from 1-2 μg/g Repeat injections of standards were also accomplished with %CVs reported in the 1-4% range.
The NIST (National Institute of Standard Technology) reference values for (±)-catechin and (±)-epicatechin in SRM (Standard Reference Material) 2384 are 245 +/-51 (μg/g) and 1220 +/-22 (μg/g) respectively with the data from this study indicating value of 254 +/-23.5 (μg/kg) and 1137 +/-68 (μg/kg) which are within the acceptable range of determinations established by NIST. While recovery studies have become a default method to assess method accuracy according to Swartz and Krull, the analysis of an established SRM is by itself a generally accepted method of validation [17]. Additionally, guidance from AOAC on methods validation indicates that spiking is not a desirable method to assess method accuracy as spiking solutions tend to be easily extractable hence the choice of the NIST standard to evaluate the method.
The sample labeled Milk Chocolate 1 is an example for a product containing very low amounts of the target analytes. With the analyte concentration in the sample extract at their lower limit of quantification and the chromatographic performance negatively affected by coextracted matrix compounds the applied method operates at its limit. Hence the sample was not included in the statistical evaluation of the method. That being said, N/A* These results were eliminated due to designation as outliers by the Q-test for outliers [16]. no issues related to complexation of polyphenols with milk reported by some researchers were seen [18]. The %CV ranged from 7-15% in this study. Laboratories used a column that satisfied the requirements of U.S. Pharmacopeia, previously described in methodology section [19]. The method was reviewed and compared with recommendations of Swartz and Krull. Finally, while the data in Table 3 may seem excessive to the casual observer, it is well within the parameters established by AOAC for another complex analyte [20].
The literature reports on the use of numerous solvents for the extraction of flavan-3-ols including mixtures of methanol, acetone, water and acid therefore the solvent combination used is within established parameters [21,22]. Furthermore, a variety of HPLC detector types have been used including UV, Diode Array, Mass Spec and fluorescence [23][24][25][26][27]. The choice of fluorescence detection is within established analytical parameters for this determination as it offers selectivity and sensitivity for these compounds with the identity of the peaks being established by the use of authentic standards.

Conclusion
The data from these studies indicate the proposed chocolate and cocoa method is suitable as an HPLC method for the determination of flavanol monomers, (±)-catechin and (±)-epicatechin in chocolate and cocoa. The method is the first such method developed by an industry group such as NCA for this purpose.