Development of HPLC for simultaneous analysis of quercetin and its degradation compound ก ก ก กก ก Saengrawee Sutthiparinyanont ( )* Dr.Aroonsri Priprem (. )** Dr. Malyn Chulasiri (. )*** Chidchanok Khamlert ( ก )**** ABSTRACT A gradient system high performance chromatography (HPLC) for simultaneous deteration of quercetin and protocatechuic acid (PA), its major degradation compound in an aqueous system, was developed for stability studies. Results obtained from the HPLC analysis was compared with antioxidant activity test by DPPH assay. The ratio of mobile phase, consisted of methanol/acetonitrile and phosphoric acid, were varied in binary and ternary systems. The optimum conditions were found in ternary mobile phase system of methanol:acetonitrile:25mm phosphoric acid with gradient program from 50:0:50 to 40:20:40, respectively. Using the developed HPLC system to analyze standard solutions of quercetin mixed with PA gave high relative coefficient (r 2 >0.99), with an approximate detection limit of 0.05 µg/ml of quercetin and 0.025 µg/ml PA, good precision (%CVs<10), and relatively high percent recovery (>87%). The developed HPLC could simultaneously use to separate quercetin and PA. Quantitative analysis by this highly sensitive method gave reliable, reproducible, precise and rapid results. Moreover, the antioxidant testing results by DPPH exhibits a good correlation with the quantitative analysis by the HPLC. This developed method is useful for further study required for pharmaceutical product development. ก (HPLC) ก (gradient) ก ก ก (PA) ก กก ก ก ก ก ก ก กก HPLC ก ก ก ก DPPH assay ก 2 3 ก ก / ก ก ก PA ก 3 ก ก 25 ก 50:0:50 40:20:40 ก PA (r 2 >99) ก ก *Student, Doctor of Philosophy Program in Research and Development in Pharmaceuticals, Faculty of Pharmaceutical Sciences, Khon Kaen University, Khon Kaen, Thailand **Associate Professor, Department of Pharmaceutical Technology, Faculty of Pharmaceutical Sciences, Khon Kaen University, Khon Kaen, Thailand ***Professor, Research & Development Division, SJI, Bangkok, Thailand ****M.D., Master of Science Program in Pharmaceutical Chemistry and Natural, Faculty of Pharmaceutical Sciences, Khon Kaen University, Khon Kaen, Thailand
0.05 ก 0.025 ก PA (%CVs<10) ก ก (>87%) ก ก ก ก ก ก DPPH assay ก กก HPLC ก ก ก ก ก ก Key Words: Quercetin, Protocatechuic acid, HPLC, Stability : ก ก ก Introduction Quercetin, 3,5,7,3,4 -pentahydroxyl flavone (Figure 1), a flavonol compound generally found in fruits, and vegetables such as onions, apple, tea, and red wines, is a potentially effective antioxidant. It has been interested for health product development for nutraceutical and pharmaceutical uses because of its potent antioxidant, antihistae, antimicrobial and anti-inflammatory properties (Formica and Regelson, 1995; Bonina et al., 1996; Ratnam et al., 2006). H (a) 6 7 8 1' A 5 4 C 3 2 2' B 6' 3' Figure 1 Structure of quercetin (a) and protocatechuic acid (b) (Makris and Rossiter, 2001) 4' 5' Figure 2 shows the degradation pathways of quercetin in the presence of hydroxyl ion. It is postulated that its degradation would affect its efficacy for health benefit. That activity may lead quercetin to the benefits in preventing serious diseases. Quercetin can be degraded via oxidative cleavage pathway under certain accelerated oxidative (b) H conditions (Makris and Rossiter 2002; Pinelo et al. 2004). To evaluate a stability profile of quercetin, which is very important for quality control of its product development and production process, a reliable quantitative analysis method is needed. Protocatechuic acid (PA) is one of major degradation product of quercetin, which has been reported to be a prooxidant (Makris and Rossiter, 2001; Cao et al., 1997). The production of PA as shown in Figure 2 would occur in aqueous rather than non-aqueous solutions. The presence of PA could be an indication of the degradation of quercetin. Most of the production and quality control of health products deals with aqueous solutions, thus, this study was intended to perform by using water as the medium for quercetin. H Quercetin xidative cleavage -2e - -2H + +2e - +2H + H H H H Phloroglucinol carboxylic acid H H H Protocatechuic acid Figure 2 xidative cleavage of quercetin. (Makris and Rossiter, 2002)
Simultaneous analysis of quercetin alone and quercetin with PA was studied in various samples such as fruits, some food and wine as it is the index. Studies by using high performance liquid chromatography (HPLC) were numerous. Quercetin alone was reported to be analyzed by various systems including isocratic mobile phase system such as Wach et al. (2007) used the mixture of HCl:methanol (60:40) and Careri et al. (2000) used the mixture of formic acid:acetronitrile (80:20). Moreover, the analysis has also been reported by using gradient mobile phase system such as mixture of phosphoric acid and methanol with gradient from 82% to 55% of phosphoric acid (Ye et al., 2002), and mixture of formic acid and acetronitrile with gradient from 50% to 65% of acetronitrile (Careri et al., 2000). The lowest limit of detection of quercetin from these reports was in the range of 64 ng/ml, and the fastest retention time of quercetin being about 5 (Ye et al., 2002). There were 2 reports on simultaneous deteration has been detered quercetin and PA being as follows. Conde et al. (1995) developed HPLC gradient system of methanol, acetronitrile and water mixture which could complete a simultaneous analysis of quercetin and PA within 21. While the system from Makris and Rossiter (2002) eluted the compound by the gradient system of acetronitrile and phosphoric acid mixture which could complete a simultaneous analysis of quercetin and PA within 90 at 40 C. However, these systems were reported for qualitative analysis with little or none information for further quantitative measurement for quality control of health products. Also, an HPLC which performed at room temperature would be preferable than the previous report with higher temperature. Quantitative analysis of a parent and its degradation compounds in the same environment is essential for the monitoring of stability, particularly compounds with a rapid oxidative degradation. Thus, there is a need to develop an HPLC which would be simultaneous analysis for quercetin and its degradation compound. This present study was aimed to develop HPLC system for simultaneous and quantitative deteration of both quercetin and PA. The developed HPLC system was then used to study the effect of solvent on immediately quercetin stability. The study was carried out by correlate of quantitative developed HPLC method and antioxidant activity testing by DPPH (2,2-diphenyl-1-picrylhydrazyl) assay in order to measure the quantitative of quercetin to scavenge stable DPPH free radical. The HPLC method would be validated and tested as a quantitative tool for detection of quercetin and PA in water. Materials and Methods 1. Materials Quercetin dihydrate, >98% purity and 3,4- dihydroxybenzoic acid (proto- catechuic acid, PA), and DPPH (2,2-diphenyl-1-picrylhydrazyl) were purchased from Sigma (St. Louis, Mo, USA). Methanol (Me) and acetronitrile (ACN) for HPLC analysis was from BDH (Poole, UK). Absolute ethanol and ortho phosphoric acid in reagent grade were from Merck (Damstadt, Germany). 2. Development of HPLC systems for the deteration of quercetin and protocatechuic acid 2.1 Concentration of phosphoric acid
The concentration of phosphoric acid was varied in order to optimize the mobile phase system, whereas the ratio of Me and phosphoric acid were fixed 60:40 ratio which modified from Ye et al. (2002). Molar concentration of phosphoric acid was varied to be 2.5, 12.5, and 25 mm. Quercetin and PA standard at 100 µg/ml prepared in ethanol, was used. The flow rate of the mobile phase was 1.0 ml/ with an injection volume at 20 µl. Quercetin and PA were detected at 294 nm by UV-visible detector. 2.2 Ratio of methanol and phosphoric acid This was an attempt to optimize the molar ratio of Me:phosphotic acid for high performance of the analysis. Ratios of Me: phosphotic acid being tested was 40:60, 50:50, 60:40, and 70:30 with isocratic system. Quercetin 100 µg/ml was injected at 20 µl and the elution was carried out with flow rate at 1.0 ml/ and detected by UV-Visible detector (294 nm). 2.3 Ratio of acetronitrile and phosphoric acid This was an attempt to optimize the molar ratio of ACN: phosphotic acid for high performance of the analysis. Ratios of ACN: phosphotic acid being tested was 40:60, 50:50, 60:40, and 70:30 with isocratic system. Quercetin standard at 100 µg/ml was injected at 20 µl and the elution was carried out with flow rate at 1.0 ml/ and detected by UV- Visible detector (294 nm). 2.4 Ternary mobile phase system Results from 2.1-2.3 were used to select a range of ratio of Me:ACN: phosphotic acid, in both isocratic and gradient systems. The ratio of Me:ACN: phosphoric acid for isocratic system was varied by 55:15:30, 45:25:30, 35:35:30, 50:10:40, 40:20:40, and 30:30:40. Then, gradient system was carried out by the elution program of system I (30:30:40 to 50:10:40), system II (50:0:50 to 40:20:40), and system III (40:20:40 to 60:0:40). 3. Validation of quercetin analysis 3.1 Standard curve and linearity Linearity of this study responded to different concentrations of the quercetin and PA standards were detered at 0.03, 0.05, 0.08, 0.1 and 4 µm. Calibration curves were linear over the investigating range when peak areas were plotted against concentrations followed a least square regression analysis. 3.2 Precision Quercetin and PA at 0.1 and 6 µg/ml were freshly prepared and repeatedly analyzed in 3 consecutive days, 5 times in a day and each analysis being 3 hours apart (five times per day with 3 hours difference). This experiment was performed 3 consecutive days. The injection volume and the flow rate were the same as described at room temperature. 3.3 Recovery The relative recovery for quercetin and PA was assessed with low and high concentrations level at 0.1 and 6 µg/ml. Each concentration of quercetin sample was spiked into various concentrations of quercetin and PA, and then was analyzed. 3.4 Limit of detection Quercetin and PA were prepared at various concentrations to detere the limit of detection of both compounds.
4. Effect of solvent on quercetin Quercetin and PA were prepared in ethanol at concentration of 20 µg/ml. Quercetin in ethanol was compared with quercetin in water. Quercetin in water system was prepared by lower concentration at 0.1 µg/ml based on its solubility. Each solution was kept in brown glass bottle for light protection at 27±1ºC. The quantity and antioxidant activity was analyzed by developed HPLC and DPPH assay, respectively. 5. Antioxidant activity testing The antioxidant activities of quercetin were assayed by modified from the DPPH assay described (Casagrande et al., 2007), the details being as follows. Quercetin and PA were separately dissolved in water and ethanol. Thereafter, the series were adjusted by methanol and finally added DPPH solution. Precisely 15 after DPPH was added, each mixture solution was measured an absorbance at 515 nm using the UV spectrophotometer. Each sample solution was prepared with 6 concentration series, each of which was subjected to linear regression analysis and the concentration of quercetin or PCA which exerts 50% antioxidant activities was evaluated as shown by this following equation: The results were reported by concentration of samples at 50% inhibition, called IC 50 % inhibition = [(Abs control Abs sample )/Abs control ] x 100 6. Statistical analysis The results reported in this work are the averages and the coefficients of variations, expressed as the percentage ratio between standard deviations and the mean values, and independent t-test for validation and comparison in stability studies. Results and Discussion 1. Development of HPLC system 1.1 Concentration of phosphoric acid Various phosphate buffer concentrations 2.5, 12.5, and 25 mm verified by HPLC, was optimized to maintain the ph value and reduced the effect of ionic strength. Figures 3 and 4 present the chromatograms of quercetin and protocatechuic acid (PA) with 3 concentrations of phosphoric acid concentration. As concentration of phosphoric acid increased from 2.5 to 25 mm the retention times of the chromatograms of quercetin and phosphoric acid increased. However, these remain good separation, suggesting that concentration of phosphoric acid did not affect the separation with retention behavior around 3.5 and 7 for PA and quercetin, respectively. It can be inferred that ionic strength is less effect for HPLC analysis in this study. In order to reduce the ionization of phenol groups, the highest phosphoric acid was selected so that the flavonols and their glycosides could be separated much better. - 25 mm - 12.5 mm - 2.5 mm Figure 3 HPLC chromatograms of quercetin using mobile phase Me:phosphoric acid with concentrations of phosphoric acid of 2.5, 12.5 and 25 mm.
- 25 mm - 12.5 mm - 2.5 mm Figure 4 HPLC chromatograms of PA using mobile phase Me:phosphoric acid with concentrations of phosphoric acid of 2.5, 12.5 and 25 mm 1.2 Effect of Me/ACN and phosphoric acid ratio Four various ratio of the mixture were carried out to detere the appropriate ratio of Me/acetronitrile (ACN) and phosphoric acid for quercetin and PA analysis rather stepwise 40:60, 50:50, 60:40, 70:30. The results are shown in Figure 5 and 6. In the system of methanol: phosphoric acid, an increase of methanol percentage gave the higher symmetric chromatograms at almost the same time around 7. While, in the system of ACN:phosphoric acid, symmetric chromatograms were occurred when reduce. The retention time was higher in the system containing lower percentage of ACN. - 50:50-60:40-70:30 Figure 5 HPLC chromatograms of quercetin using mobile phase Me:phosphoric acid with ratio of 50:50, 60:40 and 70:30 Figure 6 HPLC chromatograms of quercetin using mobile phase Me:phosphoric acid with ratio of 50:50, 60:40 and 70:30 Ternary mobile phase system (Me:ACN:phosphoric acid) was then optimized of separation condition in isocratic and gradient systems. An increasing ratio of phosphoric acid retained the chromatogram to longer time, but in contrast in the ratio of ACN (Figure7). Figure 7-70:30-60:40-50:50-40:60-55:15:30-45:25:30-35:35:30-50:10:40-40:20:40-30:30:40 HPLC chromatograms of quercetin using mobile phase Me:ACN:phosphoric acid with various isocratic ratio Finally gradient elution was carried out to ensure that each compound separate completely. Figure 8 showed chromatograms of mixed standards and the samples that could be completely separated within 15. Gradient system II [50:0:50 (3 ), 60:0:40 (3 ), 55:5:40 (3 ), 50:10:40 (3 ), 40:20:40 (3 )], revealed good separation and analysis, was then validated and used as optimized condition for stability study. Moreover, the quercetin and PA analysis using this developed condition spend
shorter time than previously study which they were eluted by UV detector at 325 nm which found chromatogram of quercetin and PA at around 21 and 5, respectively (Conde et al., 1995). In addition, quercetin and PA were detered by HPLC with UV detection at 290 nm, fixed mobile phase system at the ratio of phosphoric acid and acetronitrile (40:60), and also gradient percentage of acetronitrile from 40% to 20%. The chromatogram of quercetin and PA were found at 45 and 12, respectively (Makris and Rossiter, 2002). PA Quercetin Figure 8 HPLC chromatograms of quercetin using mobile phase Me:ACN:phosphoric acid ratio with gradient system II Me: phosphoric acid with 2. Validation of the developed HPLC system The standard range of quercetin or PA was checked and showed that linear correlations were obtained. The slope quercetin or PA was found at 0.9972 with linear equation of y = 15.024x + 17 for quercetin and y = 31.122x - 90.535 (r 2 = 0.9925) for PA. Within-day repeatability and reproducibility of the method, carried out with 0.1 and 6 µg/ml of quercetin gave the average CVs of less than 3% and 10% for 0.1 and 6 µg/ml, respectively. This indicates that the developed HPLC method is repeatable and reproducible. The relative recovery was in the range between 87 to 91%. The limit of detection (LD) for a signal-to-noise ratio of 3:1 was 0.05 µg/ml. That is reliable for use for simultaneous analysis quercetin and PA in other sample. 3. Stability of quercetin and PA in content and antioxidant activity The developed HPLC method was attempted for use in the detection of quercetin and PA in commonly used solvents, such as water and ethanol. The focus of the analysis was based on quercetin as it is the parent compound and the deteration of PA was performed as a by-product of analysis. Figure 9 shows an antioxidant activity of sample to inhibit free radical at different concentration by using DPPH assay. At fifty percentages of inhibition as IC 50 value present the potential of quercetin and PA as showed in Table 1 and32. %Inhibition 70 60 50 40 30 20 10 0 y = 14.119x - 1.4156 R 2 = 0.9995 0 1 2 3 4 5 Concentration (ug/ml) Figure 9 Inhibition curve of quercetin in ethanol to inhibit DPPH free radical by DPPH assay Changes in the quantity and antioxidant activity of an ethanol solution containing 20 µg/ml quercetin and PA during storage at ambient temperature are shown in Table 1. The results show that the quantity and antioxidant activity of both
compounds in ethanol revealed significantly difference from an initiation time since 2 h for PA and 4 h for quercetin. The change of smaller structure of PA showed the degradation was more rapid than quercetin. The decrease in quantity from an initiation time did correspond to any appreciable change in antioxidant activity. Antioxidant activity will reduce (IC 50 increase) follow by the decrease in quantity. This correlation results about quantity and antioxidant behavior of quercetin as well as PA solutions, evaluate the developed analysis method confirmed by an activity test. PA is one of degradation product of quercetin by oxidative cleavage pathway (Figure 9), it showed to exert high potential inhibit free radical almost the same activity with quercetin. Table Quantity and antioxidant activity change of quercetin and PA in ethanol solution Time (h) Quercetin Quantity (µg/ml) Quercetin PA Remark: ND = not detectable *, #, significantly Antioxidant activity by DPPH (IC 50) 0 18.6 ± 0.11 ND 3.64 ± 0.06 2 18.9 ± 0.06 ND 3.63 ± 0.04 4 18.6 ± 0.14 ND 3.84 ± 0.07 * Protocatechuic acid, PA 0 ND 18.7 ± 0.09 3.80 ± 0.13 2 ND 18.5 ± 0.06 4.03 ± 0.04 # 4 ND 18.4 ± 0.06 4.58 ± 0.09 # Water 0 0.079 ± 0.005 * ND 3.80 ± 0.04 2 0.048 ± 0.002 * ND 3.85 ± 0.07 4 0.058 ± 0.004 * ND 3.61 ± 0.05 different (p<0.05) In aqueous solution, the stability of quercetin containing 0.1 µg/ml in water based on quercetin s solubility, Changes in quantity and antioxidant activity of quercetin in water systems during storage at the ambient temperatures (27±1ºC) for 4 h are reported in Table. ver 25% decrease of quantity was observed in water after two hours of storage time. Water solution of quercetin revealed antioxidant activity almost the same as in ethanol solution. The quercetin antioxidant activity can also be emphatically affected by polarity of solvent (Pinelo et al., 2004). Some recently researches reported that the antioxidant activity of quercetin in ethanol solution was near twice higher than that observed in the organic solvent, which presents an important polar group (van der Berg et al., 1999; Pinelo et al., 2004). These data especially in water system are not in agreement with the typical behavior of quercetin, whose chemical oxidation promotes the oxidative cleavage reaction giving two major products (Figure 2) which this study used only protocatechuic acid as an index to confirm this degradation pathway. Due to the time of this study was quite short (4 h). Consequently it required to extend for longer time to clear the understanding of the behavior of quercetin degradation in aqueous system. Conclusion The developed HPLC method was validated and proved to be reproducible, reliable and accurate. It could simultaneous detere quercetin and PA from one sample by using HPLC with simple UV detector. Both compound completely eluted by ternary mobile phase of methanol, acetronitrile, and phosphoric acid with gradient program from 50:0:50 to 40:20:40 ratio. The analysis takes less than 15
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