Analytical standards production for the analysis of pomegranate anthocyanins by HPLC

Pomegranate (Punica granatum L.) is a fruit with a long medicinal history, especially due to its phenolic compounds content, such as the anthocyanins, which are reported as one of the most important natural antioxidants. The analysis of the anthocyanins by high performance liquid chromatography (HPLC) can be considered as an important tool to evaluate the quality of pomegranate juice. For research laboratories the major challenge in using HPLC for quantitative analyses is the acquisition of high purity analytical standards, since these are expensive and in some cases not even commercially available. The aim of this study was to obtain analytical standards for the qualitative and quantitative analysis of the anthocyanins from pomegranate. Five vegetable matrices (pomegranate flower, jambolan, jabuticaba, blackberry and strawberry fruits) were used to isolate each of the six anthocyanins present in pomegranate fruit, using an analytical HPLC scale with non-destructive detection, it being possible to subsequently use them as analytical standards. Furthermore, their identities were confirmed by high resolution mass spectrometry. The proposed procedure showed that it is possible to obtain analytical standards of anthocyanins with a high purity grade (98.0 to 99.9%) from natural sources, which was proved to be an economic strategy for the production of standards by laboratories according to their research requirements.


Introduction
The pomegranate (Punica granatum L.) fruit, native from Iran and endemic in the Middle East, grows in semiarid climates.In recent years there has been renewed interest in the global nutraceutical and functional benefits of this fruit, both fresh and processed (SUMNER et al., 2005).
The pomegranate fruit has some phenolic compounds such as anthocyanins (delphinidin, cyanidin and pelargonidin) in its composition, and also quercetin, phenolic acids and tannins (punicalagin).The fruit is consumed fresh or processed as juice and can be used in the food industry for the manufacture of juice beverages, soft drinks, confectionary products and also colorants (QU et al., 2011).
Due to the increased demand for healthy products, anthocyanin-rich fruits have great potential as raw materials in food formulations, acting as one of the most important natural antioxidants and being responsible for the intense red colour of pomegranate juice based products.The colour is one of the quality parameters that most promotes sensory acceptability by consumers (GIL et al., 2000;ALIGHOURCHI and BARZEGAR, 2009;BOROCHOV-NEORI et al., 2009;PATRAS et al., 2010).Thus, the analysis of anthocyanins by a reliable technique, such as HPLC, can be considered as an important tool to evaluate pomegranate juice quality.Besides the sensory-organoleptic characteristics, knowledge of the anthocyanin profile of pomegranate materials becomes important, since it allows for the identification of adulteration in these products (ZHANG et al., 2009).Thus the analysis of the anthocyanins by a reliable technique can also be considered as an important tool to evaluate pomegranate juice authenticity.
Studies on the characterization and quantification of the phytochemical and antioxidant properties of fruits have become essential and increase awareness about the different cultivars, whether natural or enhanced.Knowledge of the quality and chemical characteristics of some species provides subsidies to distinguish them from each other, and can also provide information to enable an improvement in their genetics, since the concentration and variety of types of the anthocyanins is what will determine the intensity of colouring of the various fruit cultivars (POMAR et al., 2005;ÖZGEN et al., 2009).
The major challenge in the quantitative analysis of compounds by HPLC, especially of anthocyanins, is the obtaining of analytical standards.Standardization is certainly the largest source of analytical errors, because it directly impacts the final result (KIMURA and RODRIGUEZ-AMAYA, 2002).In some countries the acquisition of high purity analytical standards usually depends on highly expensive importations, and in addition  3) with a flow rate of 1.0 mL min 1 .The column temperature was 40 °C.

Anthocyanin isolation method
The anthocyanins were isolated by liquid chromatography coupled to a Rheodyne six-channel selection valve (GOUVÊA et al., 2012).The valve was adapted to select output channels rather than possible columns, replacing the traditional fraction collector.Substances of interest were collected on elution, according to the retention time of each anthocyanin, using a selector valve commanded by Empower TM software (Waters, USA).

Concentration of the standards
For the concentration step, a Waters (USA) Sep-Pak TM C 18 cartridge previously packed with methanol was saturated with the aqueous extract of the isolated anthocyanins.The cartridge was then washed with an aqueous solution of 0.01% HCl to remove the more polar compounds present, such as sugars and phenolic acids, and the anthocyanin pigments retained were eluted with methanol.The eluted anthocyanin pigments were dried under a filtered compressed air flow, and diluted with a 5% formic acid solution in water: methanol (90:10, v/v) in a 5 mL amber volumetric flask.Aliquots of each isolated anthocyanin were injected under the same chromatographic conditions described above, and the peak areas used to check the purity.

MS/MS condition
A high resolution Waters mass spectrometer (USA) Synapt TM ESI-QTOF, with direct injection, was used to confirm the identity of the anthocyanins isolated.The MS source used was positive electrospray ionization (ESI + ) with the following conditions: source temperature at 120 °C, desolvation gas (N 2 ) delivered at 12.5 L min -1 at 500 °C, capillary exit set at 3.0 kV, sampling cone energy set at 25.0 V and extraction cone energy set at 4.0V.

Calculation of the concentrations of the standards
200 µL aliquots of the solutions obtained after concentration were collected, dried under a filtered compressed air flow and diluted with 2.0 mL of the appropriate solution for each anthocyanin, according to the molar absorptivity used (Table 4).The corresponding anthocyanin concentration of these solutions was calculated using the Beer-Lambert law from the

Sample extraction
Two grams of each freeze-dried sample were weighed into four centrifuge tubes with lids for extraction with methanol: formic acid (10:90, v/v) under sonication, following by centrifugation (BRITO et al., 2007).All the materials obtained in the supernatants after extraction were concentrated using a Büchi RE rotatory evaporator (Switzerland) at 38 °C for 4 hours.The dried extract was diluted with 4 mL of a 5% formic acid solution in water: methanol (90:10, v/v) and filtered through a hydrophilic type Millex TM membrane (0.45 µm; Merck Millipore; USA) directly into an automatic chromatograph injector vial.

HPLC-PDA evaluation of the anthocyanins
Chromatographic analysis was carried out following the methodology described by Brito et al. (2007), using a Waters (USA) Alliance TM 2695 system equipped with a Waters 2996 photodiode array detector (at 520 nm).A Thermo Scientific C 18 BDS (100 mm × 4.6 mm; 2.4 µm; USA) column was used with an injection volume of 20µL, mobile phase consisting of 5% aqueous formic acid (solvent A) and acetonitrile (solvent B) in the gradient elution mode (Table 2) with a flow rate of 1.0 mL min 1 .The column temperature was 40 °C.

Conditions for the isolation of the anthocyanins by HPLC-PDA
The anthocyanins were isolated following the methodology described by Brito et al. (2007) using a Waters (USA) Alliance TM 2695 system equipped with a Waters 2996 photodiode array detector (at 520 nm).A  The purity of the isolated anthocyanins was calculated by evaluating the peak area of each one in relation to the total area of the chromatogram.Verification of the purity of the compounds at different wavelengths ensured an accurate calculation of their concentration.
The purity of the isolated anthocyanins was also checked at 280 and 360nm, since at those wavelengths occurs the absorption by other compounds that could cause interference, such as other phenolic compounds (GIUSTI et al., 1999).Values above 90% of purity were obtained for all the six compounds evaluated at these two wavelengths.
T h e h i g h v a l u e s f o r p u r i t y o b t a i n e d ensured there were no inter fering substances that could cause bathochromic or hypsochromic effects of the ultraviolet/visible absorption spectra.
The compounds isolated in the 5% formic acid: methanol (90:10, v/v) solution showed values for concentration that were in an appropriate range to permit the construction absorbance reading obtained using a Shimadzu UV-1800 spectrophotometer (Japan).

Product evaluation
The anthocyanin profile of the different pomegranate products such as the juice and microcapsules, obtained in the laboratory using a spray drying process, were evaluated to verify the application of the method.
The six fruits chosen as anthocyanin sources made it possible to isolate these compounds by the HPLC technique with a great grade of purity at 520nm (Figure 2), which is the same wavelength that the anthocyanins are Table 4.
All the six anthocyanins isolated could be used as analytical standards, since besides the high grade of purity, the identity of each was confirmed by a reliable technique, such as high resolution mass spectrometry (Table 6).
As described by Müller et al. (2012) the use of single isolated anthocyanin standards better reflects the absolute content of these compounds in a solution than the use of just one anthocyanin to calculate all their concentrations.The same authors found higher anthocyanin contents in blueberries (Vaccinium corymbosum L.) and bilberries (Vaccinium myrtillus L.), using single isolated standards 55 , Campinas, v. 17, n. 1, p. 51-57, jan./mar. 2014 in the quantification step than by using cyanindin-3glucoside equivalents.
The proposed method is an important tool for the quality control of pomegranate drinks and other pomegranate products.The possibility of this evaluation is important not only for consumers but also for industry, since it can be used to understand and optimize some of the operational parameters that could influence the stability of the bioactive compounds, in this case, more specifically, of the anthocyanins.
Using the isolated anthocyanins and the calibration curves prepared, it was possible to evaluate the contents of these compounds in different pomegranate products such as juice and the microcapsules obtained by spray drying, as shown in Table 7.

Conclusion
The proposed procedure showed that it was possible to obtain analytical anthocyanin standards with a high grade of purity from natural sources, and proved

Table 1 .
Vegetable matrices selected for the isolation of the anthocyanins.

Table 2 .
Gradient elution mode for the evaluation of the anthocyanins.

Table 3 .
Gradient elution mode for isolation of the anthocyanins.

Table 5 .
Data for the external standard calibration curves.

Table 6 .
Identification of the anthocyanins isolated by high resolution mass spectrometry.

Table 7 .
Anthocyanin concentrations in raw pomegranate juice and microcapsules obtained by spray drying.