Introduction

Raspberry (Rubus idaeus L.) and blackberry (Rubus fruticosus L.), member of the Rosaceae family, provide delicious fruits that can be consumed fresh or as ingredient in processed products such as ice cream, jam, jelly, marmalade, purées, fruit juices, liquors, etc. In Bursa (Turkey), raspberry and blackberry are two of the most important berries, grown extensively on a commercial basis primarily for export. Most of them are sold fresh, but after harvest, their fresh fruits are highly perishable with limited postharvest life, mainly due to decay (Botrytis cinerea) and firmness loss. Therefore, fresh fruits are preserved by storage in normal and controlled atmospheres (Haffner et al. 2002) or freezing from harvest to processing (De Ancos et al. 2000b).

Berry fruits are rich in phenolic compounds contents such as ellagic acid (Rommel and Wrolstad 1993c; Häkkinen et al. 1999a, b), hydroxybenzoic acids (p-hydroxybenzoic acid, protocatechuic acid, vanillic acid, gallic acid, and syringic acid), hydroxycinnamic acids (p-coumaric acid, ferulic acid, caffeic acid; Rommel and Wrolstad 1993a; Häkkinen et al. 1999b), anthocyanins (Rommel and Wrolstad 1993a), and flavonols (quercetin, kaempferol, myricetin; Rommel and Wrolstad 1993b; Häkkinen et al. 1999a, b). The phenolic compounds in berries have been reported to have antioxidant, anticancer, anti-inflammatory, and antineurodegenerative biological properties (Seeram et al. 2006b; Seeram 2008). Ellagic acid has been reported as the main phenolic compound in raspberry and blackberry in literature (Häkkinen et al. 1999b; Mertz et al. 2007; Sellappan et al. 2002). Bioavailability and pharmacokinestics of ellagic acid from pomegranate juice in human has been studied in literature (Seeram et al. 2004). Because of the biological properties associated with berry fruits, the identification of their phytochemicals is necessary for the evaluation of raspberry and blackberry consumption on human health.

The effects of freezing and frozen storage on quality and quantity of ellagic acid, flavonols, and anthocyanin content in different cultivars of raspberries and blackberries have been studied in literature (De Ancos et al. 2000a, b; Häkkinen et al. 2000a, b; Mullen et al. 2002). However, there are no references about the affect of freezing and frozen storage on the hydroxycinnamic and hydroxybenzoic acids contents in berry fruits. The contents of phenolic compounds in berries are not only affected by genetic differences and preharvest environmental conditions but also by the degree of maturity at harvest, variety, growing area, and seasonal variations (Zadernowski et al. 2005).

Several methods and chromatographic techniques have been reported for extraction and identification of phenolic compounds in berries (Määttä-Rııhınen et al. 2004; Häkkinen et al. 1998). Phenolic extracts of plant materials are always a mixture of different classes of phenolics that are soluble in the solvent system used. Most common solvents are aqueous mixtures with methanol, ethyl acetate, and acetone. Furthermore, hydrolysis of glycoside bonds is often used in the extraction procedure, and thereby, essential information of total phenolic compounds as aglycones can be obtained (Hertog et al. 1992; Häkkinen et al. 2000a; Zafrilla et al. 2001). Various chromatographic techniques have been employed for separation and quantification of phenolic acids. High-performance liquid chromatography (HPLC) has been most widely used for both separation and quantification of phenolic compounds (Hertog et al. 1992; Häkkinen et al. 1999b, 2000a; De Ancos et al. 2000a). The use of liquid chromatography with mass spectrometry (LC–MS) detection provides useful structural information and allows for tentative compound identification when peaks have similar retention times and similar UV absorption spectra. In addition, LC–MS/MS is also useful for distinguishing compounds with identical molecular weights such as quercetin and ellagic acid (Seeram et al. 2006a; Mertz et al. 2007).

The objective of this study was to evaluate the effect of freezing and frozen storage on the concentration of individual phenolic acids (ellagic, ferulic, caffeic, p-coumaric, p-hydroxybenzoic, and protocatechuic acids) and flavonoids (quercetin, kaempferol, myricetin, and (+)-catechin) in raspberry and blackberry which they have high content of phenolic compounds. The phenolic acids and flavonoids in five raspberry and four blackberry cultivars were identified and quantified in water and methanol extracts after acid hydrolysis.

Materials and Methods

Reagents and Standards

Caffeic acid, ferulic acid, p-coumaric acid, p-hydroxybenzoic acid, and protocatechuic acid were purchased from Merck (Darmstadt, Germany); (+)-catechin hydrate, kaempferol, and quercetin hydrate were purchased from Sigma-Aldrich (St. Louis, MO, USA); and ellagic acid and myricetin were purchased from Fluka (Buchs, Switzerland). Methanol and hydrochloric acid were of analytical grade. All standard solutions were prepared in methanol (Merck, Darmstadt, Germany). HPLC grade formic acid and acetonitrile were purchased from Merck (Darmstadt, Germany).

Sampling

Raspberry fruits (R. idaeus L.) of five cultivars (Aksu Kırmızısı, Rubin, Newburgh, Holland Boduru, Heritage) and blackberry fruits (R. fruticosus L.) of four cultivars (Bursa 1, Bursa 2, Jumbo, Chester) were collected from different commercial orchards in the region of Kestel (Bursa, Turkey) and hand-harvested at commercially mature stage during the growing season of July–August 2007. The fruits (allotted in 0.5 kg lots) were packed in flat polyethylene containers. The fresh and just-frozen (frozen at a commercial frozen storage, Mevsim Foods Industry, at −35 °C in air blast freezing for 5 h) fruits were transported to the laboratory for sample preparation. Another part of polyethylene containers (0.5 kg lots) containing just-frozen fruits was stored at −22 °C for 6 months for further use.

Extraction and Hydrolysis of the Phenolic Compounds

Frozen berries were thawed at room temperature (23 °C) in dark for 30 min. Fresh, just-frozen, and frozen berries were blanched separately using water or methanol for 5 min. Blanched berries were milled in a household blender in three lots of 5 g each. Ascorbic acid (80 mg) and 10 mL of 6 mol/L HCl (final concentration 1.2 mol/L HCl) were added to crushed berries. The mixture was stirred with magnetic stirrer and kept at room temperature for 16 h in dark. The hydrolyzed samples (total volume 50 mL) were separated from the solid matrix by filtration through sheets of qualitative filter paper. A 15-mL portion of methanol extract was evaporated at 30 °C until 3 mL left using rotary evaporator. The filtrates were further passed through 0.45-μm membrane filters before LC–MS/MS analysis. Three replicates (three extractions from one container of 500 g) were analyzed for each treatment.

LC–MS/MS Conditions

LC–MS/MS analysis was performed with an Agilent 1100 LC-MSD Trap SL Model LC–MS/MS system equipped with an autosampler. Chromatographic separations were carried out using a Zorbax SB C18 (50 × 4.6 mm, i.d. 1.8 μm) column. Mobile phase consists of 1% formic acid in water (solvent A) and acetonitrile (solvent B). Gradient conditions are 0–7 min 30% B, 7–27 min 50% B, and 27–30 min 33% B; total run time is 30 min. The column was equilibrated for 10 min prior to each analysis. Flow rate was 0.2 mL/min and injection volume was 10 μL. Mass spectrometric data were acquired in positive mode with electrospray ionization source. The mass spectra were recorded in the range of m/z 100–500. Nitrogen was used both as drying gas and as nebulizing gas at flow rates of 5.00 L/min and 15.00 psi, respectively. The high-voltage capillary was held at 4,000 V. The mass spectra of each compound were obtained at extracted ion mode [M+H]+ as illustrated in Table 1. Peaks were identified on the basis of comparison of retention times and MS spectra with standards of ferulic acid, caffeic acid, p-coumaric acid, p-hydroxybenzoic acid, protocatechuic acid, ellagic acid, (+)-catechin, kaempferol, quercetin, and myricetin.

Table 1 Retention times (t R), molecular weights (M W), and fragment ions of standard phenolic compounds determined in this study
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Statistical Analysis

The data were subjected to one-way analysis of variance (ANOVA) using the Minitab software package version 14 (University of Texas, Austin, TX, USA). Statistical differences at p < 0.01 were considered to be significant and mean separations with least significant difference were applied using the MSTAT-C software package, version 2.1 (Michigan State University, Kalamazoo, MI, USA).

Results and Discussion

Effect of Extraction Methods on the Phenolic Compounds in Raspberry and Blackberry

The solvent extraction has been the most common method for extraction of diverse compounds found in fruits, including flavonoids and phenolic acids. Phenolic compounds are polar molecules; thus, the most common solvents used in the extractions are water, methanol, their mixtures with different proportions, and acidified solvents. In acidified solvent extraction, the acylated and the glycoside bonds could be destroyed that resulting in the formation of free phenolic compounds (Rommel and Wrolstad 1993a). The same phenolic compounds were identified with water extraction in raspberry and blackberry but different in quantity except for p-hydroxybenzoic acid (Figs. 1 and 2). The p-hydroxybenzoic acid was not identified in blackberry with water and methanol extractions. These results are in agreement with those reported in the literature (Sellappan et al. 2002). Similar results were also observed with methanol extraction in both fruits. In the case of individual phenolic acids, the statistically highest difference between two extractions was observed for ellagic acid and ferulic acid. Water was three times more efficient than methanol for ellagic acid and ferulic acid, whereas the efficiency of water is two times high for caffeic acid in fresh raspberry and blackberry. Regarding the extraction efficiency of the solvents, results indicated that for phenolic acids, the solvent with higher polarity extracted significantly higher amounts of compounds. These results are in agreement with those reported in the literature (De Ancos et al. 2000a; Häkkinen et al. 2000b). High extraction efficiency was obtained using solvents with 40–60% of water. Similar results were also observed for just-frozen and frozen storage periods. The phenolic compounds of p-coumaric acid and p-hydroxybenzoic acid were not identified in methanol extracts of raspberry and blackberry. Therefore, water could be an appropriate solvent for extracting hydroxycinnamic acids (p-coumaric, ferulic, and caffeic acid) and hydroxybenzoic acids (p-hydroxybenzoic) in berries. On the other hand, there is no statistical difference between water and methanol extractions with regard to quercetin, suggesting that both solvents could be used for extracting flavonols in raspberry and blackberry. Due to high efficiency of water extraction, we provided data only for water extraction for evaluating the effect of fresh, just-frozen, and frozen storage on the concentration of phenolic compounds in raspberry and blackberry.

Fig. 1
figure 1

Effect of extraction methods on the concentration of phenolic compounds in raspberry. EA ellagic acid, FA ferulic acid, CA caffeic acid, p-CA p-coumaric acid, p-HBA p-hydroxybenzoic acid, QU quercetin

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Fig. 2
figure 2

Effect of extraction methods on the concentration of phenolic compounds in blackberry. EA ellagic acid, FA ferulic acid, CA caffeic acid, p-CA p-coumaric acid, p-HBA p-hydroxybenzoic acid, QU quercetin

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Influence of Cultivar on the Phenolic Compounds in Raspberry and Blackberry

Phenolic compounds were determined in five raspberry (Aksu Kırmızısı, Newburgh, Rubin, Heritage, and Hollanda Boduru) and four blackberry (Bursa 1, Bursa 2, Chester, and Jumbo) cultivars of fresh, just-frozen, and after 6 month storage by LC–MS/MS. Ellagic acid, ferulic acid, caffeic acid, p-coumaric acid, p-hydroxybenzoic acid, and quercetin were determined in water extract of raspberry cultivars (Table 2). Five phenolic compounds were identified in water extract of blackberry: ellagic acid, ferulic acid, p-coumaric acid, caffeic acid, and quercetin (Table 3). Ellagic acid was predominant in all raspberry and blackberry cultivars and ranged from 1,828.07 ± 23.73 mg/kg fresh fruit for blackberry (Chester) to 727.90 ± 9.21 mg/kg fresh fruit for raspberry (Rubin). Ellagic acid content was found in blackberry to be higher than that previously reported in literature (Sellappan et al. 2002). This difference could be explained by different cultivars of blackberry, location, analytical method used, and extraction procedure. One-way ANOVA shows that for all phenolic compounds, there are significant differences between raspberry and blackberry cultivars (p < 0.01), and blackberry clearly differentiates from the raspberry. Ellagic acid is present as free ellagic acid, ellagitannins which are hexahydroxydiphenic acid forms esters with a sugar, and ellagic acid glycosides in berries (Rommel and Wrolstad 1993c; Häkkinen et al. 2000b; De Ancos et al. 2000a). Ellagitannins hydrolyze to glucose and hexahydroxydiphenic acid, which is spontaneously lactonized to ellagic acid. Therefore, the content of ellagic acid derivatives was analyzed as ellagic acid equivalent after acid hydrolysis. Ellagic acid content in raspberry (Heritage 1,350.36 ± 13.85 mg/kg) was higher than those other raspberry cultivars in fresh fruits (Aksu Kırmızısı 1,018.75 ± 20.16 mg/kg, Newburgh 898.51 ± 17.12 mg/kg, Hollanda Boduru 864.24 ± 3.63 mg/kg, Rubin 727.90 ± 9.21 mg/kg). Nonsignificant differences in ellagic acid content were observed in Newburgh and Hollanda Boduru fresh fruits, whereas statistically differences were found among Aksu Kırmızısı, Rubin, and Heritage fruits. The ellagic acid content was determined in four blackberry cultivars (Bursa 1, Bursa 2, Chester, and Jumbo) and Chester has the highest content of ellagic acid in fresh fruits (Tables 3). Nonsignificant differences in ellagic acid content were observed in Bursa 1 and Jumbo fresh fruits (p < 0.01), whereas statistically differences were detected among Bursa 2 and Chester fresh fruits (Table 3). The total phenolic content (considering the sum of all of the individual phenolics) ranged from 3,231 ± 41.10 mg/kg fresh fruits in raspberry (Newburgh) to 1,928.75 ± 31.47 mg/kg fresh fruits (Rubin) and ranged from 4,054.89 ± 5.79 mg/kg fresh fruits in blackberry (Chester) to 2,765.99 ± 11.08 mg/kg fresh fruits (Bursa 1). Although there is no statistical difference between Newburgh and Hollanda Boduru in ellagic acid contents, all the cultivars are significantly different in terms of total phenolic contents in raspberry, but nonsignificant differences were observed between Bursa 2 and Jumbo in blackberry. Ellagic acid was the main phenolic compound in red raspberry, forming 88% of the phenolic compounds analyzed as indicated in literature (Häkkinen et al. 1999b). Our result for ellagic acid content was much higher than previously reported for raspberry cultivars (Häkkinen et al. 1999b; De Ancos et al. 2000a). Comparison of the phenolic content of different berries from literature is difficult because of the varying analytical methods used, cultivars, growth conditions, maturity, environmental and climatic conditions, soil type, geographic locations, freezing procedure, extraction solvent, and procedure.

Table 2 Effect of freezing and frozen storage on the concentration of phenolic compounds of raspberry cultivars (milligrams per kilogram fruit)
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Table 3 Effect of freezing and frozen storage on the concentration of phenolic compounds of blackberry cultivars (milligrams per kilogram fruit)
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Three hydroxycinnamic acids (ferulic, caffeic, and p-coumaric acid) and two hydroxybenzoic acids (p-hydroxybenzoic and protocatechuic acid) were analyzed in all raspberry and blackberry cultivars. These hydroxycinnamic and hydroxybenzoic acids are present as glycosides or esters with glucose in berries (Rommel and Wrolstad 1993a). Acid hydrolysis transforms glycosylated and esterified phenolic acids into their aglycones or free phenolics. The contents of hydroxycinnamic acids varied from 90.11 ± 2.27 mg/kg fresh fruits (Hollanda Boduru) to 820.78 ± 10.65 mg/kg fresh fruits (Newburgh) in raspberry and from 112.60 ± 2.99 mg/kg fresh fruits (Bursa 1) to 877.45 ± 1.26 mg/kg fresh fruits (Chester) in blackberry. Ferulic and caffeic acid contents in fresh fruits in Newburgh are the highest, whereas the highest p-coumaric acid content was detected in fresh fruits of Aksu Kırmızısı (Table 2). These values are higher than those for fresh berries reported in the literature (Sellappan et al. 2002). p-Coumaric acid was determined to be the major phenolic compound with concentrations ranging from 287.15 ± 2.93 mg/kg fresh fruits (Bursa 1) to 877.45 ± 1.26 mg/kg fresh fruits (Chester) in blackberry. Ferulic acid was determined at concentrations between 413.82 ± 11.90 mg/kg fresh fruits (Bursa 1) and 757.69 ± 4.34 mg/kg fresh fruits (Jumbo), while caffeic acid was determined at concentrations ranging from 377.89 ± 4.40 mg/kg fresh fruits (Bursa 1) to 736.85 ± 6.93 mg/kg fresh fruits (Chester). The contents of ferulic, caffeic, and p-coumaric acids were found higher than previous results in all blackberry cultivars (Sellappan et al. 2002). These differences could be explained with extraction procedure and different cultivars examined. p-Hydroxybenzoic acid was determined in raspberry at concentrations ranging from 534.20 ± 8.95 mg/kg fresh fruits (Aksu Kırmızısı) to 233.29 ± 7.57 mg/kg fresh fruits (Heritage). Nonsignificant differences in p-hydroxybenzoic acid content were observed between Rubin and Heritage cultivars. Raspberry and blackberry cultivars did not have any detectable amounts of protocatechuic acid (data not shown). However, the protocatechuic acid was determined at 1.39%, 0.75%, and 0.44% in different raspberry juices (Rommel and Wrolstad 1993a).

The flavonols (quercetin, kaempferol, myricetin) were analyzed in five raspberry and four blackberry cultivars. The quercetin was only flavonol detected in Heritage (46.97 ± 0.32 mg/kg fresh fruits) and Hollanda Boduru (27.31 ± 0.27 mg/kg fresh fruits; Table 2). Aksu Kırmızısı, Newburgh, and Rubin cultivars did not have any detectable amount of quercetin. Kaempferol and myricetin were also not detected in all raspberry cultivars (data not shown). The quercetin was also only flavonol detected in all blackberry cultivars with concentrations ranged from 56.78 ± 2.32 mg/kg fresh fruits (Bursa 1) to 74.69 ± 2.71 mg/kg fresh fruits (Chester; Table 3). Flavonols are present as glycosides in berries (Rommel and Wrolstad 1993b), and they are more soluble in water (Hertog et al. 1992). The glycosides can be hydrolyzed to aglycones with strong acids. As can be seen from the results of raspberry and blackberry, the content of quercetin was higher in all cultivars. The quercetin was not detected in some of blackberry cultivars (Sellappan et al. 2002; Siriwoharn and Wrolstad 2004), but Amakura et al. was detected 6.21 mg/100 g of quercetin in blackberry after acid hydrolysis (Amakura et al. 2000). The content of quercetin in raspberry was determined to be higher than that previously reported by Häkkinen et al. (1999a) and Mullen et al. (2002). Differences in flavonol contents may partly be due to different cultivars or to methodological differences. Also, environmental factors (e.g., light, temperature, and soil nutrients) may influence flavonol concentration in plants (Häkkinen et al. 1999a). Discrepancies may be also due to differences in maturity stage of the analyzed fruit and applied different extraction and analyzed procedure. Trace concentrations of flavan-3-ols (+)-catechin and (−)-epicatechin were determined in red raspberry juice (Rommel and Wrolstad 1993a), but we did not detect flavan-3-ols (+)-catechin (data not shown) in all raspberry and blackberry cultivars.

Effect of Storage on the Phenolic Compounds in Raspberry and Blackberry

The concentration of ellagic acid was quantified in raspberries and blackberries of fresh, just-frozen, and after 6 months frozen storage. The results showed that the freezing treatment affected the ellagic acid concentration in raspberry and blackberry cultivars (Figs. 3 and 4). In raspberries and blackberries, markedly lower ellagic acid levels were measured after 6 months of storage at −22 °C than fresh raspberries and blackberries. Significant changes in phenolic compounds contents due to long-term frozen storage were observed in raspberry and blackberry. The percentage loss of phenolic compounds depended on the cultivars studied. Greater degradation of ellagic acid was shown in two cultivars of raspberry (Aksu Kırmızısı and Heritage) 49% and 50%, respectively (Fig. 3). Ellagic acid was the most stable among the phenolic compounds in blackberry. After 6 months at −22 °C, 95%, 44%, 68%, and 82% of the start levels remained in the blackberry cultivars of Bursa 1, Bursa 2, Chester, and Jumbo, respectively. Previously, a decrease in ellagic acid content during storage has been reported for raspberry (De Ancos et al. 2000a; Häkkinen et al. 2000b; Zafrilla et al. 2001). The most probable explanation for this could be the affect of freezing procedure, frozen storage, and the cultivar considered. On the other hand, variation in the content of ellagic acid in different cultivars could be observed due to environmental factors (e.g., light, temperature, and soil nutrients) and methodological differences The losses of ellagic acid in raspberries during the frozen storage at −22 °C could be related to free ellagic acid that may act as metal chelating compound with metallic cations, e.g., Mg+2 and Ca+2, ability to react with free radicals (Rommel and Wrolstad 1993c; Häkkinen et al. 2000b), and also the enzyme of polyphenol oxidase (PPO) linked to the cellular wall, which could release with cellular disruption in berry fruits (De Ancos et al. 2000a). The enzyme of PPO releases by the broken cells and the presence of molecular oxygen oxidizes the polyphenolics to quinones. On the other hand, the ellagic acid content in frozen samples was slightly higher than that in just-frozen samples. This slightly changes in ellagic acid content in just-frozen raspberries could be explained by the degradation of ellagitannins, release of hexahydroxydiphenic acid from ellagitannins, which is transformed to ellagic acid. The other reason of high ellagic acid content in raspberry is the degradation of the cell structures of berries, which ellagic acid is strongly linked to the cell walls. Therefore, ellagic acid could be extracted easily from frozen berries during the frozen storage (Rommel and Wrolstad 1993c; De Ancos et al. 2000a; Häkkinen et al. 2000b; Zafrilla et al. 2001, Mullen et al. 2002). Ellagic acid content in just frozen of Bursa 1 significantly decreased (Table 3). However, ellagic acid content of the frozen fruit (Bursa 1), which was stored for 6 months at −22 °C, significantly increased. On the other hand, gradual decrease was observed in the content of ellagic acid in Chester, Jumbo, and Bursa 2 cultivars during frozen storage. Nonsignificant differences in ellagic acid content were observed in Aksu Kırmızısı and Newburgh fruits during just-frozen storage, whereas statistically differences were observed among just-frozen and frozen storage in Rubin, Heritage, and Hollanda Boduru fruits (Table 2).

Fig. 3
figure 3

Efect of frozen storage on the phenolic compounds of five raspberry cultivars

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Fig. 4
figure 4

Efect of frozen storage on the phenolic compounds of four blackberry cultivars

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The ferulic acid, caffeic acid, and p-coumaric acid content decreased in just-frozen raspberry fruits (Aksu Kırmızısı, Newburgh, Rubin, and Heritage; Table 2). However, ferulic acid, caffeic acid, and p-coumaric acid contents of the frozen fruits (Aksu Kırmızısı, Newburgh, Rubin, and Heritage), which were stored 6 months at −22 °C, significantly increased. On the other hand, caffeic acid content in frozen sample was higher (326.93 ± 4.69 mg/kg) than just-frozen sample (218.01 ± 4.41 mg/kg) in Bursa 1 (Table 3). Frozen storage had no significant impact on the content of caffeic acid in Aksu Kırmızısı (92%) and Heritage (97%) and p-coumaric acid in Rubin (91%; Fig. 3). p-Coumaric acid was only determined and quantified in all blackberry cultivars. p-Coumaric acid content in fresh fruit in Bursa 1 was the lowest. Frozen treatment differently affected to the p-coumaric acid content in all blackberries. Therefore, a gradual decrease was observed in the content of p-coumaric acid only in Bursa 2 during frozen storage. p-Coumaric acid content of just-frozen blackberry fruits (Bursa 1, Chester, and Jumbo) was lower than frozen fruits (Bursa 1, Chester, and Jumbo). The increase in contents of ferulic, caffeic, and p-coumaric acids in berries could be explained by either their better stability compared to other phenolics or due to their better release from the matrix. The possible another explanations for this result could be the degradation of complex phenolic structures such as tannins and flavonoids into phenolic acids (Bunea et al. 2008) or some of simple phenolics can increase as a result of breakdown of supramolecular structures containing phenolic groups (Bunea et al. 2008) and also due to degradation of cell structure. However, frozen storage may negatively affect the concentration of phenolics while the enzyme of polyphenol oxidase is released by the broken cells, which is responsible for the presence of molecular oxygen that oxidizes polyphenolics to quinones. p-Hydroxybenzoic acid was only detected in raspberry (Table 2), and a gradual decrease was observed in the content of p-hydroxybenzoic acid in all fresh, just-frozen, and frozen raspberries in descending order. However, protocatechuic acid was not detected in all raspberry and blackberry cultivars (data not shown). Statistically differences were observed in p-hydroxybenzoic acid content in Aksu Kırmızısı, Newburgh, and Heritage fruits during just-frozen and frozen storage, whereas nonsignificant differences were observed during frozen storage in Rubin and Hollanda Boduru.

Quercetin was determined only in two cultivars (Heritage and Hollanda Boduru) of raspberry in fresh, just-frozen, and frozen storages (Table 2), whereas it was determined in all blackberry cultivars (Table 3). The quercetin content was significantly higher in 6 months frozen storage (57.34 ± 2.65 mg/kg) than fresh fruit samples (46.97 ± 0.32 mg/kg) and just-frozen storage (38.82 ± 0.37 mg/kg) in Heritage. Statistically differences were observed in quercetin content in Heritage and Hollanda Boduru during just-frozen and frozen storages. The reason for low content of quercetin in just-frozen storage could be due to the low content of vitamin C in berries (Häkkinen et al. 2000a). High level of vitamin C can inhibit the degradation of quercetin as an antioxidant during frozen storage (Häkkinen et al. 2000a). The other reason might be due to breakdown of quercetin or enzymatic and oxidative reactions during frozen storage. The freezing process can cause small changes in the vitamin C content in raspberries. The losses of vitamin C were observed during long-term frozen storage in different raspberry cultivars (De Ancos et al. 2000a; Häkkinen et al. 2000a; Mullen et al. 2002). On the other hand, a gradual increase in quercetin content was observed in Hollanda Boduru during frozen storage (Table 2). Previous studies showed significant increase or decrease in quercetin contents during frozen storage in raspberry cultivars (Häkkinen et al. 2000a; Mullen et al. 2002). We observed that the quercetin content in Heritage and Hollanda Boduru increased during 6 months frozen storage (Table 2). The explanation for these observations could be due to degradation of cell structure of fruit; therefore, the quercetin could be easily hydrolyzed and extracted during frozen storage. The difference in the quercetin content between our results and literature (Häkkinen et al. 1999a, 2000a) could be explained by extraction methods, cultivars, or growth conditions of the fruits. Environmental factors (e.g., light, temperature, and soil nutrients) have also influence on the flavonol concentrations in fruits. The highest quercetin content was found in Chester cultivar of blackberry. The quercetin content decreases during just-frozen storage only in Bursa 2. On the other hand, a significant increase was observed in quercetin content during just-frozen storage in Bursa 1, Chester, and Jumbo (Table 3). The quercetin content was higher in just-frozen storage than fresh fruit samples and 6 months frozen storage in Bursa 1 and Jumbo.

Validation of Analytical Method

The validation of the quantitative determination of phenolic compounds in raspberry and blackberry sample was performed by limits of detection (LOD; 3s/m), limits of quantification (LOQ; 10s/m), and recovery (percent) of each phenolic compound (Tables 4 and 5), where s is the sample standard deviation for the replicates and m is the slope of the calibration curve. LOD ranged from 0.002 to 0.050 mg/kg and LOQ ranged from 0.010 to 0.090 mg/kg for ten phenolic compounds. The extraction efficiency of the phenolic standards of ellagic acid, ferulic acid, caffeic acid, p-coumaric acid, p-hydroxybenzoic acid, and quercetin from raspberry and blackberry samples were evaluated by spiking the mixture of phenolic compounds to samples and extracted using water in acidic medium (Table 5). The recovery study was carried out only for the phenolic compounds identified by LC–MS. The mean percentage recoveries ranged from 70 ± 1% (ellagic acid) to 91 ± 2% (p-coumaric acid) for raspberry and ranged from 65 ± 3% (p-hydroxybenzoic acid) to 92 ± 1% (ferulic acid) for blackberry. Similar recoveries were obtained for raspberry and blackberry except for ellagic acid and p-hydroxybenzoic acid. The least recovery was obtained as 70 ± 1% for ellagic acid in raspberry, but 91 ± 1% in blackberry. On the contrary, 65 ± 3% of recovery was obtained for p-hydroxybenzoic acid in blackberry, but 88 ± 1% in raspberry. All other recoveries are in experimental error range. The influence of some berry matrices was detrimental for the analysis of some phenolic acids. This may be due to their chemical reactions as metal chelators or copigmentation reactions with other phenolics, for example, anthocyanidins (Häkkinen et al. 1999a). In the calculation of the final results, the recoveries of the pure standards were not taken into account.

Table 4 Validation parameters of phenolic compounds
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Table 5 Recovery (percent) of the phenolic compounds in raspberry and blackberry samples
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Conclusions

Ellagic acid was the main phenolic compound in raspberry and blackberry and the least degraded after frozen storage for 6 months in raspberry. The varietal differences in the phenolic compound contents were larger among the blackberry cultivars (from 1,828.07 to 56.78 mg/kg fresh fruit) than among the raspberry cultivars (1,350.36 to 27.31 mg/kg fresh fruit). Blackberry cultivars contained higher level of ellagic acid, caffeic acid, p-coumaric, and quercetin than raspberry cultivars, whereas raspberry cultivars were higher in ferulic acid content and also raspberry contained an addition of p-hydroxybenzoic acid. The results show that although a decrease in phenolic compounds was observed with just frozen, an increase in the contents of phenolic acids with long-term frozen storage was observed and this could affect the bioavailability of these compounds. The frozen storage more quickly degraded the p-hydroxybenzoic acid (79%) than ferulic (30%), caffeic acid (8%), and p-coumaric acids (12%) in raspberry (Aksu Kırmızısı). Hollanda Boduru was more affected by the long-term frozen storage and remained as the cultivar with the lowest phenolic compounds after 6 months frozen storage at −22 °C. p-Coumaric acid content was not significantly changed during frozen storage in Chester and Jumbo, but its content was reduced about to 40% of fresh fruit after frozen storage of 6 months. Although the contents of quercetin are the lowest in raspberry and blackberry, the frozen storage does not significantly affect its content in long-term storage. Blackberry has higher phenolic compounds and less affected by long-term storage than raspberry. Therefore, fresh fruits of raspberry could serve as potential natural sources of phenolic compounds and freshly picked raspberry should be consumed for human health. On the other hand, blackberry could be more suitable berry for freezing and long-term storage with regard to the phenolic compounds studied in this work.