High Performance Liquid Chromatography (HPLC) with the Pre-Column Fluorescence Derivatization is a method in which the separation and ultra-sensitive detection of a substances occur. This sensitive method simultaneously detects eight amino acids (AAs); glycine, alanine, serine, glutamic Acid, arginine, tyrosine, phenylalanine, and tryptophan. These AAs has been developed and validated in different species of tea. Moreover, the method was validated and successfully applied to determine the AAs content in tea species (the tea products (green, black, oolong, and white) were used to determine the AAS), according to the official guidelines. Considerable variations were observed based on the tea quality and type. In summary, this study, determination of the amino acids content in tea species, can be used for the online monitoring of the tea productions process to determine the grade quality, adulterations and detect the AAs intake.
Role of Amino Acid
Amino acid (AAs) contains amine groups, carboxylic acid group, also a side chain that specify to each amino acid. There are twenty elements present in amino acids that are found in proteins, Glycine, Alanine, Valine Isoleucine, Leucine, Proline, Methionine, Phenylalanine, Tyrosine, Tryptophan, Serine, Cysteine, Threonine, Asparagine, Glutamine, Aspartic acid, Glutamic acid, Histidine, Lysine, and Arginine. This acid plays a major role to regulate different process that are related to gene expression, also the proteins that use to mediate messenger RNA (mRNA) translation. Amino acids can be classified in different side chains. A basic amino acid has a functional group in its side chain, while an acidic functional group in its side chain is called an acidic amino acid. Chemical properties present in amino acids determines the biological activity of the protein. Such AAs can be obtained in some drinks and foods that contain high number of free essentials and non-essential AAs
Two thirds of the world’s population consumed different tea made from the processed leaf of Camellia Sinensis. Plant teas are mostly made up of water, and they begin to lose water when they are removed in the plant. Some important compounds in fresh tea leaves are polyphenols, amino acids, enzymes, pigments, carbohydrates, methylxanthines, minerals and many volatile flavor and aroma compounds. In the recent years, the health benefits of tea can help the prevention of cancer and cardiovascular diseases. But, adding tea to the diet may cause other serious health concerns. Tea helps to dehydrate the body it can provide a rich and flavorsome source of water.
Principles in Liquid Chromatography
The term ‘Chromatography’ refers to passing through a mixture dissolved in a mobile phase, usually a liquid or a gas, through a stationary phase.
Separation process based on distribution between two phases are the principle of liquid chromatography wherein the sample components are pushed by the liquid which percolates a solid stationary phase. Liquid- liquid chromatography (LLC) and other older techniques have been replaced by the Bonded phase chromatography (BPC) and Liquid solid chromatography (LSC) that has a largest impact in the liquid chromatography. The separation of sample components may be achieved both in low- and high-pressure system.
Liquid-liquid chromatography commonly called ‘Partition’ is one of the powerful techniques for separation and analysis of a wide variety of sample types. In liquid chromatography, chemically bonded organic stationary phases which is the most widely used supports.
Bonded phase chromatography is most commonly used for LC mode and it’s a stationary phase chemically bonded to support that is used for the separation of a sample.
Liquid solid chromatography has a major mechanism of retention that is adsorption and also utilizes a solid stationary phase.
Material and Method
A list of AAs found and studied are high purity of alanine, arginine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.
Methanol, tetrahydrofuran, Sodium hydroxide, phosphoric acid and acetonitrile were of HPLC grade. Sodium acetate trihydrate, sodium borate, hydrochloric acid (HCl), sodium hydroxide, and glacial acetic acid were of reagent grade.
A set of derivatizing reagent contains AccQ-Fluor reagent powder (6-Aminoquinolyl -N-hydroxysuccinimidyl Carbamate), AccQ-Fluor borate buffer and AccQ-Fluor reagent diluents and AccQ-Tag Eluent A. Also, a derivatization reagent of ophtalaldehyde (OPA) (98%) and 3-mercaptopropionic acid (3MPA) (99%).
All chemicals were used as received without further purification. Deionized water was used for the preparation of standard solutions, buffers, dilutions, and eluent systems. All mobile phases and solutions were filtered.
A stock solution of each amino acid (AA) was separately prepared, by dissolving an accurately weighed amount of each AA, with its final concentration. The prepared solutions were stored frozen until further use. Further dilutions of the stock solutions were made for the appropriate calibration concentrations, followed by filtration through 0.45 μm filters.
The derivatization reagent was freshly prepared by mixing OPA and 3MPA. A standard volume of 5 mL of this derivatization reagent mixture was added to the standard AA solutions or the extract of each tea sample, allowed to react for 5 min at ambient temperature, filtered through 0.45 μm filters. An aliquot was immediately injected through the HPLC system. The blank samples were prepared without AAs were also treated in the same way.
The HPLC Analysis of the researchers are as follows:
HPLC system (Agilent Technology-Series 1200) equipped with a fluorescence detector and composed of a binary pump with a column temperature regulator was used. Chromatography Chemstation soft- ware (Agilent, German) was used for data processing. The HPLC peaks were generated upon the injection of 20 μL of each AAs after their pre- column derivatization and filtration
The fluorescence detection setup was set at an excitation wavelength (λEx) of 340 nm with an emission wavelength (λEm) at 450 nm. The HPLC separations were performed at 30 °C, using Agilent Zorbax Exclipe XDB- C18 column (5 μm, 250 mm × 4.6 mm i.d., Agilent Technologies, U.S.A.). A binary gradient mobile phase (eluent A & B) was used at a flow rate of 1.5 mL per minute. Eluent A was composed of 20 mM acetate buffer (pH 7.6) containing 3.0% Tetrahydrofuran (v/v) and eluent B was a mixture of 100 mM acetate buffer: acetonitrile: methanol in the volumetric ratio of 1:2:2. The gradient elution timeline was programmed at different time intervals with overall record chromato- gram time of 15 min. Between analysis, the HPLC column was pre- equilibrated with isocratic elution mode of eluent A as a mobile phase.
Tea Products for Analysis
Using eleven commercial tea samples were investigated in this study.
These samples include five black teas from different origins i.e. one Silane origin (Sri Lanka) (Sam 1), one Indian origin (Sam 2), and other three different Chinese grades (Sam 3 to 5). The other tea types were of two green tea samples (Sam 6 & 7), two oolong teas (Sam 8 & 9), and two white teas (Sam 10 & 11), each type is differed in the cultivating origins of Sri Lanka and China, respectively. A constant 2.5 g of each individual tea sample was grounded into homogenous powder using a grinder, and stored in the desiccators at room temperature before infusion. As previously reported for sample preparation, tea powder of 0.30 g was extracted by 20 mL distilled water at 80 °C for 20 min (staring well during the incubation) and then cooled to room temperature. Subsequently, the infusion was collected by centrifuging the solid matter at 12,000 rpm for 10 min, make the volume again up to 20 mL with distilled water, and filtered through a
0.45 μm nylon filter membrane. Following this, 1 mL of the obtained filtrate (as the infused tea sample) was diluted with water to 10 mL and used for pre-column reaction with 5 mL derivatization reagents and proceed for HPLC measurement. Only in the case of Gly quantification and due to the low level of this AA in tea infusion no dilution was made for the filtrate. The timeline for each individual step of the process preparation from tea powder infusion to the HPLC injection was set to 60 min as the total experiment time.
Result and Discussion
The eight major AAs where been reported found in the samples of tea species. These AAs were glycine, alanine, serine, glutamic Acid, arginine, tyrosine, phenylalanine, and tryptophan (Table 1).
Derivatization of AAs were performed with the certain reagents (the mentioned reagents above) and were converted into fluorescent species for the fluorescence detector detect it easily. This process was performed due to the lack of fluorophore groups that is required in the fluorescence detection.
Amongst such reagents is OPA that has been reported to react with AAs in alkaline medium, in the presence of other mercaptan compounds such as 3MPA.
With the help of this procedure, the non-fluorescent AAs were converted into highly fluorescent species that is according to the data has a fluorescence signal at ~450 nm. The reaction was relatively rapid as it occurs simultaneously within 5 min at the room temperature with high sensitivity.
To initiate the reaction with the secondary amine groups 3-mercaptopropionic were combined with OPA, also to increase the product stability.
The optimization of chromatographic conditions is required for the optimum separation of each amino acid from their mixture. Different mobile phases were checked and ration were tested. According to the result, using an isocratic system of eluent A, the optimum chromatographic conditions were obtained.
Moreover, to remove all the remaining chemicals between each analysis, the HPLC column was pre-equilibrated with isocratic elution mode of eluent A. Such elution system and conditions produced a well-separated chromatogram for the eight AAs, with no peak over- lapping as the column resolution factors and within a relatively short time.
(Table 3: A comparison between the sensitivity of the proposed method and previous chromatographic methods with fluorescence detection [13,18] for the analysis of AAs in tea.)
The possible procedure to use for the analysis of AAs was approved for quality and control laboratories using the guidelines of International Conference on Harmonization (ICH), in linear range, the limit of detection (LOD), the limit of quantitation (LOQ), the accuracy, precision and robustness.
Precision, accuracy, and robustness
Precision and accuracy of the studied method were known using three measurements for each AAs, at different levels of concentration, within the linear ranges. They are represented as the recovery percentage of ± relative standard deviation (RSD), Table S3. The acquire results exhibits an enough precision as the RSD values did not surpass 3% of the recovery percentage. Additionally, the agreement within the measurements of each concentration and closeness to the mean specify the good accuracy of this method. The robustness of the method was approximated by investigating how the small change in the mobile phase composition on the analytical performance of the method affects. Hence, some little change did not have an impact to the result (the percentage of recovery) and shows an acceptable performance of this method for application in laboratories.
Application of the proposed method to detect AAs in tea products
To detect the investigated AAs in the eleven tea products, the researchers extend the method developed in this study. The preliminary study was performed by adding an internal standard of mixture and detecting the chromatographic peaks .
The resulting chromatogram showed a combined peak for each AA (spiked and extracted from the tea product) at the same specified retention time of the respective standard AA that was mentioned in Table 2.
The study was further applied to detect the AA content in each tea product by spiking 0.5, 1.0 and 5.0 nmol/L of each infused sample and following the described procedure of pre-column derivatization with (OPA:3MPA, 3:1) and chromatographic separation. The amount of each AA in the investigated tea product was determined as illustrated in Table 4.
The results show that the highest in all tea products that was analyzed and compared with the other AAs was the level of glutamic acid. Also, for green tea, the highest amongst all other were the level of glutamic acid and tryptophan while glycine was the lowest detected AA in such tea products.
Black tea products showed a relatively similar AA amount to that of green tea, the only difference was the quality of it.
In contrast, the oolong tea shows higher levels in Gly even though this AA shows relatively very low content in all of the other types of tea studied. In addition, the levels of serine, alanine, and tyrosine were markedly higher in white tea, compared to all other green or black tea which could indicate the absence of fermentation (oxidation) in white tea.
These results reveal that the free AAs content in tea is highly dependent on the tea type, grade, and quality.
Rapid analytical method for the determination of AAs content in tea species or tea products has been developed and validated, for the application in quality control of each laboratories. The method exploits the selective chemical reaction between the non-fluorescent AAs with o- phthaldialdehyde (OPA) and 3-mercaptopropionic (3MPA) to convert them into fluorescent compounds (isoindoles).
The derivatization method wass performed and optimized and the fluorescent isoindoles exhibited fluorescence signal. Also, High performance liquid chromatography (HPLC) was used to isolate each amino acids fr0om the mixture.
The overall method was highly sensitive to detect the eight studied AAs with a limit of detection as low as 0.32 nmol/L. The results showed marked variations in the AA content based on the tea product, grade, quality and type.
Such results indicated that glutamic acid and serine are the most abundant AAs in all tea types expect white tea which contains relatively low levels of serine, alanine, and tyrosine. In summary, this method is beneficial for the tea-producers to monitor the process of on-line production to control the type and quality of the final product. In addition, this method helps consumers to select their fa- vorite tea taste and monitor the AA intake of each product.