ANALYSIS OF FERULIC ACID IN ARABICA COFFEE BEAN ( COFFEA ARABICA L.) USING SOLID PHASE EXTRACTION–HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

Objective: The purpose of this research is quantitatively analyzing of ferulic acid in the Arabica coffee bean extract from three samples with different regions (Garut, Pangalengan, Tasikmalaya, West Java Indonesia) using Solid Phase Extraction-High Performance Liquid Chromatography (SPE-HPLC) method which is validated. Methods: Results: The results showed that the precision of retention was 8.853 min, correlation coefficient (R) was 0.9996, and the recovery was 96.909%. The quantitative analysis of ferulic acid content in the extract of coffee from thus, samples of three different regions were 0.0385%, 0.0169% and 0.0076%, respectively. The analysis method used reversed phase HPLC with an Enduro C 18 G (250 mm × 4.6 mm) column and detector UV 312 nm, with a mobile phase of methanol and water containing 1% (v/v) of acetic acid (42:58) at a flow rate of 1.0 ml/min and validation method was examined in linearity, Limit of Detection (LOD), Limit of Quantification (LOQ), precision, and accuracy. Conclusion: The analytical method was meet the validation criteria. Ferulic acid levels results from the extraction of the digestion process and pretreatment methods of Solid Phase Extraction (SPE) is 0.0385% from Garut area, 0.0169% from Pangalengan area and 0.0076% from Tasikmalaya area.


INTRODUCTION
Coffee has many active compounds, one of them is polyphenols such as ferulic acid [4]. Ferulic acid is one type of phenolic acids which are found in the cell wall components, leaves and seeds of plants. The ferulic acid is produced from the metabolism of phenylalanine and tyrosine. This acid has an important role for plants because of its ability to stop the radical chain reaction. In 100 grams of arabica coffee contained ferulic acid as much as 23 to 120 mg [4][5][6][7].
Ferulic acid has potential as anticancer, antioxidant, and antiaging. Research shows that ferulic acid can lower blood glucose and cholesterol and triglyceride levels. It also has therapeutic effects in diabetes, cardiovascular disease, anti-inflammatory, antimicrobial activity, antiallergic, antiplatelet, and antiviral [5,6,8].
The objective of this research was to develop a novel RP-HPLC method for simultaneous estimation of ferulic acid in Arabica coffee beans from three regions of West Jawa district (Pangalengan, Tasikmalaya and Garut) using Solid Phase Extraction-High Performance Liquid Chromatography (HPLC) method. This research can be used as a reference of ferulic acid level which can be developed into a product that is useful in pharmaceutical development.

Plant material
Arabica coffee beans (Coffea arabica L.) obtained from 3 of the province at West Java, Indonesia is Pangalengan, Tasikmalaya and Garut (Specimen number.: 20/HB/10/2014). Pangalengan is a city with significant rainfall, its climate is mild and generally warm and temperate. Its temperature averages 16.1 °C. Tasikmalaya is the city with the average daily temperature are mildly varied, it ranges from 20 ° to 34 °C at lowland areas and 18 ° to 22 °C at the upland areas. While, Garut has a cool but tropical climate, with an average temperature of 24 °C.

Collection, determination, and processing
The collection of material was obtained from Pangalengan, Tasikmalaya and Garut, West Jawa Indonesia. Determination was done at Herbarium Laboratory of Plant Taxonomy Department of Biology, Faculty of Mathematics and Natural Sciences, Padjadjaran University. Processing of materials was done using grinder the beans into powder form to increase the surface area before extraction.

Extraction
A total of 20 grams of seeds that have been grinded then extracted using 250 ml aquabidestilata at a temperature of 40-50 °C for 30 min with a magnetic stirrer [24].

HPLC conditions
HPLC system to be used is to use Enduro C-18 column (250 mm × 4.6 mm) and 312 nm UV detector, with a mobile phase of methanol and aquabidestilata containing 1% acetic acid with a ratio of 42:58 at a flow rate of 1 ml/min.

Mobile phase preparation
The mobile phase that is used was methanol and aquabidestilata containing 1% acetic acid.

Standard solution preparation
A total of 25 mg of ferulic acid standards are dissolved in 25 ml flask with aquabidestilata to have stock solution concentration on 1000 ppm range.

Sample solution preparation
The samples that is used to do pretreatment prior to the method Solid Phase Extraction (SPE). Stationary phase SPE cartridge is activated by the stationary phase (Bakerbond-C18) passed with 9 ml of methanol and 9 ml of water is slowly using a vacuum. The stationary phase should remain wet. 1 ml sample is introduced to a SPE cartridge and the vacuum is turned on, make sure the stationary phase remains wet. The filtrate should not be removed, but re-enter the filtrate into the same SPE tube, turn on the vacuum, this time drag all the fluids and collect them in different test tubes. Stream the air through an SPE tube for 3-5 min until the stationary phase is dry. After that, the ferulic acid was eluted twice with 12 ml of absolute ethanol. SPE sample results are then dried and then reconstituted with 24 ml aquabidest.

Collection, determination, and processing material
The material test is arabica coffee that obtained from the area Pangalengan, Garut and Tasikmalaya. These three regions are the largest coffee-producing center in West Java. Determination Results that have been conducted at the Laboratory of Plant Taxonomy, Department of Biology, Faculty of Mathematics and Natural Sciences, University of Padjadjaran showed that the plants that is used in the study is the Arabica coffee plant (Coffea arabica L.) Family Rubiaceae. Processing plants is done by grinding the beans before extraction which aims to increase the surface in order to gain efficiency and rate of extraction of soluble components [25].

Extraction results
Extractions were performed using digestion method by stirring continuously at a temperature higher than room temperature, generally at a temperature of 40-50 °C [25]. Selection of this method is intended to contain very small ferulic acid content in coffee beans can be extracted. Digestion is done by soaking 20 grams of powdered coffee beans into 250 ml of solvent aquabidest then stirred continuously using a magnetic stirrer for 30 min. The digestion process maintained below temperature 50 °C because Ferulic acid has stability at 50°C for approximately 20 h. The reason for using Akuabides solvent because it is a solvent that readily available and cheap, non-volatile, stable, non-toxic and are commonly used general public to consume coffee. The Extraction results with digestion is liquid mass (liquid extract) with organoleptic that greenish-yellow solution to brownish green and typical aroma of coffee beans. Each liquid extract that obtained from Pangalengan, Garut and Tasikmalaya are 150 ml, 160 ml, and 160 ml.

Phytochemical screening results
The phytochemical screening method was carried out with specific reagents for each group in order to know the phytochemical compounds contained in the extract of coffee beans [26,27] as shown in table 1.
Phytochemical screening results showed that samples of arabica coffee beans of the three regions containing alkaloid metabolites, flavonoids, polyphenols, moniterpenoids, steroids, triterpenoids and quinones. This shows that differences in location of growth do not affect the presence of the womb metabolites in arabica coffee beans.

Optimization of HPLC conditions
Optimization of HPLC conditions performed by compare several compositions of mobile phase. The choices composition used for analysis is based on some parameters. System suitability test is performed to ensure the system is running effectively and well. The parameters used include retention time, column efficiency (N), tailing factor and column efficiency (HETP) [15]. The result shown at table 2. From these results the selection of the conditions used was methanol and aquabidest containing 1% acetic acid (42:58) with a flow rate of 1 ml/min resulted in more efficient retention time. The retention time of 8.853 min was chosen to ensure that the ferulic acid standard was not covered by the irresistible retention time of the solute at 2.18 min. In addition, this mobile phase composition has the smallest HETP value and the largest amount of theoretical chips in which the efficient column is capable of producing a narrow peak and separates the analyte well. Value plates will be even greater if the size of the longer column, this indicates that the separation process that occurs the better [16] .
Results chromatogram of the methanol mobile phase composition and water which contain 1% acetic acid (42:58) at a wavelength of 312 nm, flow rate 1 ml/min using a column of C-18 is as follows. The peak of ferulic acid is at a retention time of 8.853 min. as shown in fig. 1.
While HETP inversely proportional to N so that the column that provides the value of N large and small HETP value can separate the components in a sample better [28].

Result of ferulic acid analysis in arabica coffee bean extract
The determination of ferulic acid levels was performed by injecting the pre-treated sample with SPE into HPLC columns under optimized and validated conditions. The results of the analysis of the chromatograms obtained in the form that can then be calculated from the levels seen widely Area Under the Curve (AUC) at a retention time ferulic acid.
Based on the data in table 5, it can be seen that the levels of ferulic acid in coffee bean extract that comes from Garut more than Tasikmalaya and Pangalengan. Ferulic acid levels in coffee bean extract is 0.0385% from Garut area, 0.0169% from Pangalengan area and 0.0076% from Tasikmalaya area.
From table 5 showed that the levels of ferulic acid in coffee bean extract from Garut have the highest levels compared to Pangalengan and Tasikmalaya. This Content difference can be caused due to differences location of the three arabica coffee beans grow. Ideally, Arabica beans grow at temperatures between 15-24 °C and receive rainfall about 15-30 cm per year. In addition, the growth of coffee under the auspices of trees is one of the basic principles in coffee planting system traditionally because it can reduce excessive light [33]. So that differences in climatic conditions, rainfall in Garut, Pangalengan, and Tasikmalaya will affect the quality and quantity of the content in arabica coffee beans.

CONCLUSION
The results showed that ferulic acid analysis method by HPLC (High Performance Liquid Chromatography) at the optimum condition that is using Enduro C-18 column with a mobile phase ratio of methanol: aqua-bidestilat that contains 1% acetic acid 42:58, the maximum wavelength of 312 nm, and a flow rate of 1 ml/min meets the requirements of the validation parameter. Ferulic acid levels results from the extraction of the digestion process, and pretreatment methods of Solid Phase Extraction (SPE) is 0.0385% from Garut area, 0.0169% from Pangalengan area and 0.0076% from Tasikmalaya area.
Further research is needed to optimize the analysis of ferulic acid content in coffee beans from various regions in Indonesia so that it can be used in the development of pharmaceutical preparations of the Arabica coffee bean extract (Coffea arabica L.).