Abstract
In this research, a novel method was developed for the matrix solid phase dispersion (MSPD) followed by high-performance liquid chromatography (HPLC) quantification of four marker constituents (vitamin C, gallic acid, rutin, and ellagic acid) in the freeze-dried pomegranate fruit juice. Various MSPD parameters like type of dispersant, sample–dispersant ratio, solvents, its volume, and time of extraction have been optimized after many trials. Furthermore, HPLC method has been developed and optimized for the analysis of all four components. The HPLC separation was achieved using a 250 × 4.6 mm column, particle size of 5 μm, C18 reverse phase column, with a mobile phase consisting of acetonitrile and 0.05% H3PO4, in gradient elution mode with a mobile phase flow rate of 1 mL/min, using ultraviolet (UV)–visible detection at 254 nm. All calibration curves showed good linear regression (r2 ≥ 0.9925) within test ranges. The extraction recoveries of the marker constituents analyzed by MSPD methods were found as ranging from 97.5% to 103.5%. From comparing the chromatograms, validation data and other parameters like time, labor, and feasibility, we found that MSPD technique was most suitable for the analysis as compared to conventional liquid–liquid extraction technique.
Introduction
Pomegranate is one of commonly used fruits which is cultivated in a wide geographical area from Europe (Spain, Italy, Turkey, Greece) and Southern Asia (India, Pakistan, Bangladesh) to middle east regions (Saudi Arabia, Egypt, Yemen, Syria). The presence of high content of polyphenols makes pomegranate a powerful astringent and anti-inflammatory agent which can be applied in the treatment for trauma hemorrhage, ulcers, infections, and disorders of the digestive tract such as diarrhea and dysentery [1, 2]. Studies also proved the antioxidant and anti-atherosclerotic properties which also attributed to its high content of polyphenols in its free and bound gallotannins, anthocyanins, and other flavonoids [3–5]. The antioxidant activity of pomegranate is three times stronger than many other dietary sources of polyphenols, such as green tea and Emblica officinalis [6]. The daily intake of pomegranate reduces the risk of atherosclerosis, cancer, diabetes, and neurodegenerative disorders and promotes health [7, 8].
Varieties of pomegranate fruit species and their value added products are now available for consumers. A survey conducted recently on pomegranate fruit juices for its “polyphenol contents” showed surprisingly that there was some of the product which even contains no traces of ellagitannins [9]. It became an extremely important issue to have some quality control methods to ensure the authenticity.
The sample extraction procedure for the HPLC analysis of herbal compounds was conventionally done by liquid–liquid and solid-phase extraction. An attractive alternative for liquid–solid extraction procedures for the purification of crude extracts maybe matrix solid-phase dispersion (MSPD) [10–14]. The MSPD extraction technique can successfully replace the conventional classical liquid–liquid and solid-phase extractions and effectively overcome most of the complications associated with conventional extraction procedures. The cost, labor, and time can be saved when compared to conventional extraction procedures by reducing cleanup steps and quantity of solvents. MSPD method found compatible due to less interference of impurity peaks in the chromatographic run [15].
In MSPD technique, samples are triturated with the solid support materials like silica/modified silica and this mixture of disrupted sample and sorbent transferred to a suitable column depending upon the quantity of samples and the components is eluated by suitable solvents depending on the polarity of targeted compound [15].
In recent years, MSPD became popular for extraction of herbal samples because of the simplicity in operation and efficiency. This extraction technique also helps to avoid unwanted components form the sample which results in achieving good chromatographic separation. Literature review proved that no such works have been reported on the MSPD extraction and analysis of these components in pomegranate yet.
One of the main concerns among the analysis of fruit juices and beverages is their vulnerability to thermal degradation which will eventually turn out to destruction of many pharmacologically active components. Therefore, there is need to develop and adopt non-thermal techniques (extraction and analysis) for the analysis of fruit juices and beverages in order to ensure the maximum recovery of the components. Moreover, the complexity nature of the analytes and their low presence in the sample makes the “sample extraction technique” significant aspect of herbal sample analysis [16]. In this study, a novel MSPD method was developed and applied for the extraction of targeted compounds from the freeze-dried extract of pomegranate fruit juice and its detection and quantification by HPLC–ultraviolet (UV).
Materials and Methods
Commercially available pomegranate fruits were purchased from supermarkets in Al-Kharj, Riyadh. Pomegranate is opened by scoring it with a knife and breaking it open; the seeds are separated from the peel and internal white pulp membranes. The fruit juice was prepared using Avance collection juicer HR1871/00 (Philips). The juice was then freeze-dried in a Millrock LD85 tray type freeze dryer (Millrock Technology, USA) at −40 °C for 24 h and stored in airtight container at −18 °C until extraction, since it is highly hygroscopic in nature. HPLC-grade acetonitrile and methanol (E. Merck, Darmstadt, Germany) were used for the analysis. Deionized water was purified by Milli-Q system (Millipore, Bedford, MA, USA). Ortho-phosphoric acid (H3PO4) 88% was purchased from Fisher Scientific Company (UK). All the standards (<99% purity) were purchased from Sigma-Aldrich, St. Louis, MO, USA. Silica gel (230–400 mesh), aluminum oxide (150 mesh), C18 silica gel, and end capped (230–400 mesh) and solid-phase extraction tubes (SPE) were purchased from Sigma-Aldrich, St. Louis, MO, USA.
Matrix Solid-phase Dispersion
About 0.5 g of the freeze-dried pomegranate juice was mixed with equal proportion of sorbent (C18 silica gel) and triturated thoroughly in a mortar. The sample–sorbent mixture was transferred to a polypropylene SPE tube with filter disc on both sides after compression injection syringe plunger and attached with SPE vacuum manifold. The components were eluted with 5 mL of 70% methanol. The procedure was repeated for 3 times to ensure the complete recovery of the components (total 15 mL of solvent). The fractions were collected, pooled, and evaporated under nitrogen flow. The dried extract was then reconstituted in 2 mL of methanol and filtered through 0.45 μm syringe filter prior injecting to HPLC.
Liquid–liquid Extraction
One gram of sample (in triplicate) was taken in a 50 mL conical flask, and 25 mL of solvent (70 % methanol) was added and mixed well. It was sonicated for 40 min at room temperature and filtered using Whatman filter paper no. 4 (the extraction solvent and time of sonication was optimized and selected after several trials for maximum recovery of all four components). The filtrate obtained was transferred to a separating funnel (100 mL volume) and extracted with 25 mL of hexane to remove undesired nonpolar compounds. Aqueous methanolic extract was dried using rotavapor below 40 °C, and the residues obtained were reconstituted in 10 mL volumetric flask and make up the volume. All the sample solutions were filtered through 0.22 μm syringe filter before injecting.
HPLC Instrument Condition
The analysis was carried out on a Waters Alliance e2695 separating module (Waters Co., MA, USA) using UV–visible detector (Waters 2998) with autosampler and column oven. The instrument was controlled by use of Empower software installed with equipment for data collection and acquisition. Compounds were separated on a C18 reverse-phase column (25 × 4.6 mm; particle size, 5 μm; Merck, Germany) maintained at room temperature. The mobile phase used was consisting of acetonitrile and 0.1% orthophosphoric (H3PO4) acid in gradient elution method starting acetonitrile percentage 10 to 100 in 20 min. The flow rate was 1.0 mL/min; the column was maintained at room temperature.
Validation of HPLC Method
Linearity was assessed with the aid of serially diluted calibration solutions of all standards. Calibration graphs were plotted on the basis of triplicate analysis of each calibration solutions by using peak area against concentration. To evaluate the accuracy, the pre-analyzed samples were spiked with standard at three different known concentration levels, i.e., 50%, 100%, and 150%, and the mixtures were reanalyzed by the proposed method. Precision of the method was determined by carrying out the intra-day and inter-day variation tests. Inter-day and intra-day precisions were done by preparing and applying three different concentrations of standards in triplicate six times a day and similarly on six different days, respectively. The relative standard deviation (RSD) was taken as a measure of precision. Robustness of the proposed method was determined in two different ways, i.e., by changing the mobile phase flow rate (1 ± 0.2 mL/min) and detecting wavelength (254 ± 4 nm). The % RSD of the experiment was calculated to assess the robustness of the method. The limit of detection (LOD) and limit of quantification (LOQ) were determined on the basis of signal-to-noise ratio. The LOD is considered to be least concentration of the analyte that gives a measurable response (signal to noise ratio of 3). The LOQ is the smallest concentration of the analyte, which gives response that can be accurately quantified (signal to noise ratio of 10).
Results
Optimization of MSPD and LSE–SPE Procedure
Different MSPD parameters have been optimized in order to gain the maximum extraction efficiency. For this, several experiment conditions were examined like extraction time, solid support, sorbent–sample ratio, eluting solvent, volume of solvent etc. In the case of the investigation for the suitable sorbent for MSPD, the most reported and common is the use of silica-based C18 sorbent due to its good mechanical strength and reproducibility. Out of three different sorbents tried (C18, silica, and alumina), it has found that the maximum yield was achieved in C18 solid support (alumina ˂ silica ˂ C18).
Another important aspect which helps in achieving efficient extraction is the selection of suitable solvent. Aqueous methanol was supposed to be a suitable solvent for the extraction of phenolic and flavanoid compounds than 100% methanol [17–20]. Different ratios of aqueous methanol (100, 80:20, 70:30, 60:40, v/v) were investigated. It was observed that the concentrations of the components were remarkably affected by the variations in the composition of methanol–water. Since optimization of extraction procedure was aimed to maximize the recovery of all for components, 70% of aqueous methanol found to be optimum for the extraction of all the components using MSPD.
The sorbent and the sample ratio were also studied for the appropriate concentration. Out of the different ratios of sample–sorbent (1:1, 1:2: 1:4, 1:5) tested, the appropriate one is found as 1:1. When the concentration of sample is too low or too high, it was found that the recovery is going down to less than 80%. In higher concentration, it was found that the sample got chocked in the column itself, which leads to lot of solvent consumption and time. It also found decreasing the recovery percentage. The MSPD extraction efficiency can affect by the extraction time. The rate of extraction of all four components found increasing with the time and reached the saturation level at 45 min.
Optimization of Optimization of Chromatographic Conditions
Gradient elution pattern was selected for the HPLC analysis since the polarity range of the components found was very narrow, which helped in the efficient separation of the components with good resolution. The mobile phase was selected using different compositions of methanol–water and acetonitrile–water with some modifiers including orthophosphoric acid, formic acid, acetic acid, phosphate buffer, and acetate buffer with different pH values adjusted using triethyl amine and ammonia, which were investigated under different gradient elution modes. After many trials, excellent separation of all components was achieved on acetonitrile and 0.1% orthophosphoric (H3PO4) acid in gradient elution method, starting acetonitrile percentage of 10 to 100 in 20 min. The representative HPLC–photodiode array (PDA) chromatograms of all the reference compounds were shown in Figure 1. The UV spectra of each analyte were determined independently to get the λmax of all nine components. In order to detect all the components simultaneously with good sensitivity, 254 nm was selected as the detecting wavelength for the analysis.
HPLC chromatogram of four reference standards at 254 nm
Citation: Acta Chromatographica Acta Chromatographica 30, 3; 10.1556/1326.2017.00053
Method Validation
The method validation was carried out as per International Conference on Harmonization (ICH) guidelines [21]. In linearity, standard stock solution of four reference standards was prepared by dissolving them in methanol (1000 μg/mL). Working curves were constructed by plotting the peak areas of analytes versus their concentrations. Corresponding linear regression equations and correlation coefficients are listed in Table 1. The recovery of the method was determined by spiking previously analyzed samples with standards which are having known purity. The proposed method was spiked in three different levels and found to be within the limit of 97.9–103.5%. The values of recovery % and % RSD are listed in Table 2. In intermediate precision, intra-day and inter-day precisions were carried out. Intra-day and inter-day precisions were done by preparing and applying three different concentrations of standard in triplicate six times a day and similarly on six different days, respectively. The low values of % RSD indicate the reproducibility and adaptability of the proposed method for the routine analysis of these markers. Assay for each analysis was calculated and reported in terms of % RSD in Table 3. Robustness study was completed to ensure continued performance of the methods in diverse analytical environments. The small and deliberate changes made in the analytical parameters (flow rate and wave length) did not find affecting the chromatography visually. The parameters like retention time and peak areas were found unharmed during the changes, which proved that the method is robust enough to work with different analytical environments (Table 4). The LOD of the four compounds investigated was estimated as the minimum concentration of the compounds needed to produce signals that were at least three times stronger than the noise signal (S/N ≥ 3). The LOD for the all four analytes were less than 0.07 μg/mL, which indicated that the analytical method was acceptable with sufficient sensitivity. LOQ of method was determined as the concentration which gives the signal ten times stronger than the noise (S/N ≥ 10). The LOQ for the all four analytes was less than 0.095 μg/mL, which indicated that the analytical method was acceptable with sufficient sensitivity (Table 1).
Analytical performances of the developed method (n = 3)
| Compound | Linearity range (µg/mL) | Correlation coefficient | LOD (µg/mL) | LOQ (µg/mL) |
|---|---|---|---|---|
| Vitamin C | 0.10–500 | 0.9985 | 0.07 | 0.095 |
| Gallic acid | 10–1000 | 0.9965 | 4.50 | 9.50 |
| Rutin | 1–500 | 0.9925 | 0.45 | 0.85 |
| Ellagic acid | 1–500 | 0.9986 | 0.40 | 0.95 |
Recovery analysis of the method (n = 3)
| Compound | Amount of drug spiked (µg/mL) | % of recovered drug (µg/mL) | % RSD |
|---|---|---|---|
| Vitamin C | 37.5 | 99.2 | 0.21 |
| 50 | 101.5 | 1.21 | |
| 62.5 | 98.0 | 0.58 | |
| Gallic acid | 142.5 | 97.5 | 0.55 |
| 190 | 102.5 | 2.58 | |
| 237.5 | 98.8 | 1.56 | |
| Rutin | 12.1 | 99.1 | 0.68 |
| 16.2 | 97.9 | 0.33 | |
| 20.2 | 103.5 | 0.97 | |
| Ellagic acid | 78 | 100.6 | 2.08 |
| 104 | 100.7 | 1.31 | |
| 130 | 98.4 | 1.11 |
Inter- and intra-day precisions of the developed method (n = 3)
| Compounds | Retention time (RSD) % | Peak area RSD (%) | ||
|---|---|---|---|---|
| Intra-day | Inter-day | Intra-day | Inter-day | |
| Vitamin C | 2.88 | 1.1 | 0.75 | 0.25 |
| 3.1 | 1.8 | 0.26 | 0.86 | |
| 1.6 | 2.22 | 0.65 | 0.25 | |
| Gallic acid | 0.92 | 0.99 | 2.11 | 0.58 |
| 0.22 | 0.28 | 0.68 | 0.64 | |
| 0.32 | 2.6 | 0.28 | 1.2 | |
| Rutin | 1.2 | 1.5 | 0.35 | 1.3 |
| 0.98 | 1.26 | 0.88 | 1.25 | |
| 0.88 | 2.01 | 1.22 | 1.55 | |
| Ellagic acid | 0.38 | 0.88 | 0.36 | 0.54 |
| 0.22 | 0.76 | 0.58 | 0.91 | |
| 0.58 | 0.55 | 0.78 | 0.12 | |
Robustness of the method by changing flow rate of the mobile phase and detection wavelength (n = 3)
| Vitamin C | Gallic acid | Rutin | Ellagic acid | ||||||
|---|---|---|---|---|---|---|---|---|---|
| Retention time (RSD) % | Peak area RSD (%) | Retention time (RSD) % | Peak area RSD (%) | Retention time (RSD) % | Peak area RSD (%) | Retention time (RSD) % | Peak area RSD (%) | ||
| Flow rate (mL/min) | 0.8 | 0.77 | 1.9 | 1.20 | 1.81 | 0.66 | 0.66 | 0.72 | 0.47 |
| 1 | 0.64 | 0.98 | 0.58 | 2.11 | 0.49 | 0.49 | 2.11 | 0.69 | |
| 1.2 | 0.66 | 0.41 | 1.50 | 0.52 | 0.52 | 0.25 | 1.41 | 0.24 | |
| Detection wavelength (nm) | 250 | 0.22 | 0.78 | 0.78 | 0.22 | 0.71 | 1.15 | 0.22 | |
| 254 | 0.14 | 0.47 | 0.56 | 0.56 | 0.47 | 0.77 | 1.20 | 0.58 | |
| 258 | 1.22 | 0.55 | 1.60 | 1.60 | 0.25 | 1.50 | 0.38 | 0.39 | |
Discussion
The MSPD extraction technique and HPLC method has been developed and optimized for the simultaneous extraction and quantification of selected marker components in the freeze-dried extract of pomegranate juice. All the samples were analyzed according to the optimized extraction procedure described earlier in the section. For the first time, MSPD extraction technique was used for the analysis of major compounds from pomegranate freeze-dried juice samples. The MSPD extraction method was found good alternative for conventional liquid–liquid extraction technique. The values of recovery % (97.9–103.5%) ensured the good extraction efficiency of the method. The main advantage of the method includes the simplicity of extraction procedure as well as simultaneous detection and quantification of bioactive components in a single chromatographic run. Moreover, the MSPD method resulted in the successful extraction of a higher percentage of compounds than traditional extraction, with fewer impurities in a shorter extraction time.
Application in Real Samples
The freeze-dried juice of pomegranate was extracted using the optimized MSPD and liquid–liquid extraction technique and analyzed by developed HPLC method. The content of vitamin C, gallic acid, rutin, and ellagic acid found by both the methods were given Table 5. The MSPD extraction and cleanup method found helps in removing impurity peaks in the chromatographic run. Moreover, the use of suitable elution solvent has brought down the impurity percentage in the chromatograms, comparing the same with chromatogram obtained from liquid–liquid extraction, which leads to accurate and reproducible analysis (Figure 2).
Content of four active compounds in freeze-dried pomegranate juice by HPLC
| Freeze-dried pomegranate juice | Mean contents (% w/w) ± SD (n = 3) | |||
|---|---|---|---|---|
| Vitamin C | Gallic acid | Rutin | Ellagic acid | |
| Liquid-liquid extraction | ||||
| 0.011 ± 0.001 | 0.091 ± 0.008 | 0.0055 ± 0.0001 | 0.055 ± 0.0011 | |
| MSPD extraction | 0.025 ± 0.002 | 0.095 ± 0.011 | 0.0081 ± 0.0001 | 0.052 ± 0.0016 |
HPLC chromatogram of freeze-dried pomegranate juice by using (A) liquid–liquid extraction and (B) MSPD extraction techniques at 254 nm
Citation: Acta Chromatographica Acta Chromatographica 30, 3; 10.1556/1326.2017.00053
Conclusion
The method described a novel technique which can successfully employ for the extraction and quantification of some bioactive components (vitamin C, gallic acid, rutin, and ellagic acid) from freeze-dried pomegranate fruit juice. The study describes all the optimized parameters for the efficient extraction including sample pretreatment method, selection of appropriate sorbent, solvent, time etc. The HPLC chromatographic conditions like mobile phase, detection wavelength, run time etc. also been optimized. Moreover, our study is the first of its kind on the MSPD extraction and analysis of selected compounds in freeze-dried pomegranate juice. The results of spiking recovery and other validation experiments suggest that the method proposed is rugged and efficient enough for the routine analysis. The suggested method was attractive over conventional methods on less consumption of toxic solvents, cost, labor, time, and simplicity in operation.
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