Kinetics of Thermal Degradation of the Anthocyanins Delphinidin-3-rutinoside and Malvidin-3-glucoside

The kinetics of the thermal degradation of the two anthocyanins delphinidin-3-rutinoside and malvidin-3-glucoside at 78, 88, 98 and 108 °C, depending on the pH and the type of solvent has been investigated. The first order reaction of delphinidin-3-rutinoside and malvidin-3-glucoside degradation has been established. The rate constants (K ± 10^?5s^?1), the activation parameters, factor Q₁₀ and the half-decay period have been calculated. The rate of delphinidin-3-rutinoside degradation is higher than that of malvidin-3-glucoside.     

Identification and characterisation of anthocyanins in the antioxidant activity-containing fraction of Liriope platyphylla fruits

The objective of this study was to investigate antioxidant activities and anthocyanin profiles in the fruits of Liriope platyphylla, where these are considered functional substances in Korea. The acidic methanol extract of this species exhibited potent antioxidant activities, showing 83.9% DPPH scavenging activity and 92.5% ABTS scavenging activity at a concentration of 0.5 mg/ml. Moreover, anthocyanins were identified by reversed-phase C₁₈ column chromatography, NMR spectroscopy, and HPLC-DAD-ESI/MS analysis. Seven anthocyanins were characterised, including delphinidin-3-O-glucoside (1), delphinidin-3-O-rutinoside (2), cyanidin-3-O-glucoside (3), petunidin-3-O-glucoside (4), petunidin-3-O-rutinoside (5), malvidin-3-O-glucoside (6), and malvidin-3-O-rutinoside (7). Among these, petunidin-3-O-rutinoside (5) (7302.2 μg/g) and malvidin-3-O-rutinoside (7) (5776.1 μg/g) were the predominant anthocyanins, whereas the least prevalent anthocyanin was found to be cyanidin-3-O-glucoside (3) (64.9 μg/g). Therefore, our results suggest that strong antioxidant activities of the acidic methanol extract of L. platyphylla fruits are correlated with high anthocyanin contents, particularly the petunidin-3-O-rutinoside (5) and malvidin-3-O-rutinoside (7).     

Resolution and analysis of total phenolic constituents of grape pigment

A procedure for the analysis of plant phenolics is described which allows the quantitative isolation of the high-molecular-weight tannin fraction, the separation of acylated anthocyanins from other anthocyanins, and the further partial separation of the monomeric polyphenols by adsorptive partition within each zone. The system used is a column of Sephadex G-25 gel prepared in an aqueous acetone medium at low pH and eluted with the same solvent. By this means, an extract of the pigment from a single berry of Vitis vinifera cv. Shiraz has been separated into three distinct pigment fractions, the first of which, a red tannin pigment, was excluded from the gel. The second and third bands contained acylated anthocyanin 3-glucosides and anthocyanin 3-glucosides respectively, the components of each band being eluted in order of their decreasing R_F in normal paper chromatography. Other phenolics of corresponding R_F were found in each of these latter fractions. Pigment composition, expressed in terms of the three fractions, was calculated by use of mean extinction values. The red tannin pigment is the major fraction of total extractable pigment in the grape, although it is only a minor contributor to total colour of extracts.     

Modified dispersive liquid–liquid microextraction followed by high-performance liquid chromatography for the determination of clenbuterol in swine urine

A modified dispersive liquid–liquid microextraction (DLLME) technique combined with an HPLC-UV procedure was developed for the extraction and determination of clenbuterol in swine urine. The modification involved the selection of methyl tert-butyl ether (MTBE) as the dispersive solvent, which had a low solubility in aqueous samples, playing the part of dispersion with the help of violent shaking. MTBE improved the partition of clenbuterol into the extractant, and helped the formation of phase separation. Various factors affecting the extraction efficiency including selection of the organic extractant and the dispersive solvent, the volume of extractant and dispersive solvent, salt concentration, NaOH concentration and centrifugation time were evaluated and optimised. Under the optimal conditions, precision, linearity (correlation coefficient, r ^2?=?0.996 over the concentration range of 10–1000?ng?ml^?1), detection limit (2.4?ng?ml^?1) and enrichment factor of 55 were obtained. The modification to the DLLME made it suitable for analytes with pronounced solubility, especially when the compounds are highly polar and thus more difficult to extract effectively by DLLME. The procedure was suitable for the fast screening of clenbuterol residue in swine urine.     

LC-MS/MS multi-analyte method for mycotoxin determination in food supplements

A multi-analyte method for the liquid chromatography-tandem mass spectrometric determination of mycotoxins in food supplements is presented. The analytes included A and B trichothecenes (nivalenol, deoxynivalenol, 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol, neosolaniol, fusarenon-X, diacetoxyscirpenol, HT-2 toxin and T-2 toxin), aflatoxins (aflatoxin-B₁, aflatoxin-B₂, aflatoxin-G₁ and aflatoxin-G₂), Alternaria toxins (alternariol, alternariol methyl ether and altenuene), fumonisins (fumonisin-B₁, fumonisin-B₂ and fumonisin-B₃), ochratoxin A, zearalenone, beauvericin and sterigmatocystin. Optimization of the simultaneous extraction of these toxins and the sample pretreatment procedure, as well as method validation were performed on maca (Lepidium meyenii) food supplements. The results indicated that the solvent mixture ethyl acetate/formic acid (95:5, v/v) was the best compromise for the extraction of the analytes from food supplements. Liquid–liquid partition with n-hexane was applied as partial clean-up step to remove excess of co-extracted non-polar components. Further clean-up was performed on Oasis HLB? cartridges. Samples were analysed using an Acquity UPLC system coupled to a Micromass Quattro Micro triple quadrupole mass spectrometer equipped with an electrospray interface operated in the positive-ion mode. Limits of detection and quantification were in the range of 0.3–30 ng g^?1 and 1–100 ng g^?1, respectively. Recovery yields were above 60% for most of the analytes, except for nivalenol, sterigmatocystine and the fumonisins. The method showed good precision and trueness. Analysis of different food supplements such as soy (Glycine max) isoflavones, St John's wort ( Hypericum perforatum), garlic (Allium sativum), Ginkgo biloba, and black radish (Raphanus niger) demonstrated the general applicability of the method. Due to different matrix effects observed in different food supplement samples, the standard addition approach was applied to perform correct quantitative analysis. In 56 out of 62 samples analysed, none of the 23 mycotoxins investigated was detected. Positive samples contained at least one of the toxins fumonisin-B₁, fumonisin-B₂, fumonisin-B₃ and ochratoxin A.     

Optimization of Extraction of Bioactive Compounds from Black Carrot Using Response Surface Methodology (RSM)

In this study, optimum conditions for the extraction of black carrot anthocyanins were determined by response surface methodology. Central composite design of extraction factors (pH 2.5–6.5, temperature 4–72 °C, solvent/solid ratio 5:1–25:1 v/w, ethanol/water ratio 0:100–100:0?v/v) was generated as two replicates. Total phenolic content, total monomeric anthocyanin content, polymeric color, total antioxidant activity, and anthocyanin composition determined by high-performance liquid chromatography were used as responses. Except for color analysis, higher temperature, solid/solvent ratio, and ethanol concentration were observed to increase the extraction yield. However, polymeric color results were found to have minimum values at lower pH and solid/solvent ratio, lower or moderate temperature, and higher ethanol concentration. Optimum extraction conditions were found as follows: 50 °C, pH 3.5, solvent solid ratio 10:1 (v/w), and ethanol/water ratio 75:25 (v/v) when all responses were considered. The validation of the optimum conditions for black carrot extraction was performed at specified values.     

Identification of anthocyanins and distribution of flavonoids in tamarillo fruit (Cyphomandra betaceae (Cav.) Sendt.)

The major tamarillo (Cyphomandra betaceae) anthocyanin pigments were isolated and identified as pelargonidin-3-rutinoside, pelargonidin-3-glucoside, cyanidin-3-rutinoside, cyanidin-3-glucoside, delphinidin-3-rutinoside and delphinidin-3-glucoside. The intense purple-coloured jelly surrounding the seeds contained the greatest concentration of anthocyanins, delphinidin-3-rutinoside being the major pigment. Flavones, flavonols and leucoanthocyanins were also present in this material. The yellow-coloured flesh contained flavones and low concentration of anthocyanins. The major anthocyanin of the skins is cyanidin-3-rutinoside; flavones and leucoanthocyanins are also present. It is suggested that the presence of leucoanthocyanins in pigment extracts induced degradation of anthocyanins during isolation and purification.     

Ionic liquid as an effective solvent for cell wall deconstructing through astaxanthin extraction from Haematococcus pluvialis

Haematococcus pluvialis is a proficient source of natural antioxidant astaxanthin. However, the efficient extraction of astaxanthin from this microalga remains a great challenge due to the presence of the tough and non-hydrolysable cell walls. In this study, ionic liquid (IL) pretreatment was used for deconstruction of cell wall method. Imidazolium-based ILs exhibited higher cell disruption capability than pyridinium-based and ammonium-based ILs. After the ILs determination, 1-butyl-3-methylimidazolium chloride ([Bmim] Cl) was the most efficient method for cell wall deconstruction that leads to the highest astaxanthin extractability. More than 80% astaxanthin was extracted from H. pluvialis under mild conditions (pretreatment with 40% IL aqueous solution at 35 °C, followed by methanolic extraction at 50 °C). In addition, [Bmim] Cl showed the excellent recyclability, and negligible loss of astaxanthin during IL pretreatment was observed. The present work demonstrates that the combination of IL pretreatment and organic solvent extraction was an energy efficient and eco-friendly process for the astaxanthin recovery from H. pluvialis.     

Effect of ionic liquid-containing system on betulinic acid production from betulin biotransformation by cultured <Emphasis Type="Italic">Armillaria luteo</Emphasis>-<Emphasis Type="Italic">virens</Emphasis> Sacc cells

The aim of this study is to develop an ionic liquid-containing system for efficient production of betulinic acid by cultured cells Armillaria luteo-virens Sacc ZJUQH100-6. Several parameters affecting betulinic acid formation in the IL-containing system were investigated. The addition of [EMIM][BF₄] in hexane-containing reaction medium gave rise to better betulinic acid formation in comparison with other ILs used. The optimal concentration of IL in IL-containing co-solvent system is 50% (v/v). As a co-substrate, butanol is found to be useful for intracellular betulin-28-monooxygenase synthesis during the whole phase. The concentrations of substrate and resting cells have been found to exert a significant effect on betulinic acid. Moreover, the reaction time in this IL-containing system was less than that in the conventional one. The effect of the constructed IL-containing system on cell membrane structure was comparatively observed. Under the developed IL-containing system, the highest yield of product observed was 11.14% at 18 h, higher than that in monophase aqueous one (P < 0.05), whereas the activity of monooxygenase showed the same variation as betulinic acid formation.     

Fractioned High Pressure Extraction of Anthocyanins from Elderberry (<Emphasis Type="Italic">Sambucus nigra</Emphasis> L.) Pomace

Fractionated high pressure extractions from dry and in natura elderberry pomace were performed in order to obtain anthocyanin rich extracts. Experiments were carried out using CO₂ supercritical fluid extraction followed by enhanced solvent extraction (ESE) with CO₂/EtOH–H₂O mixtures (1–100%, v/v), to obtain anthocyanin rich fractions in the second step, at 313 K and ~20 MPa. Higher extract yields, anthocyanin contents and antioxidant activities occurred by the presence of water, both in the raw material and in the solvent mixture. The CO₂ dissolved in the ESE solvent mixture favored either anthocyanin contents or antioxidant activities, which were not directly related. Comparing to the literature data for elderberries and grapes, these fractions had higher anthocyanins contents. From these results, an added economical value to this agroindustrial residue is proposed, using solvents and techniques “generally regarded as safe” in the food and pharmaceutical industries.     

Phenolic Analyses of Haskap Berries (Lonicera caerulea L.): Spectrophotometry Versus High Performance Liquid Chromatography

Haskap berries (Lonicera caerulea L.) are known for their high phenolics, anthocyanins, and antioxidant potential. The data on the phenolic profile of these fruits are lacking. In this study, the phenolic profiles of three haskap varieties; tundra, berry blue, and indigo gem grown in Nova Scotia, Canada were investigated for the first time using spectrophotometery and high-performance liquid chromatography. Berries were analyzed for total phenolic content/total reducing capacity, total anthocyanin content, and antioxidant potential (2,2-diphenyl-1-picrylhydrazyl radical scavenging activity). The total reducing capacity, total anthocyanin content, and 2,2-diphenyl-1-picrylhydrazyl values were 6.17–8.42 mg gallic acid equivalents/gram fresh weight, 4.49–6.97 mg cyanidin-3-glucoside equivalents/gram fresh weight, and 78.70–89.55%, respectively. The extracts were analyzed by reversed-phase diode array detector–high-performance liquid chromatography through a gradient elution using Synergi 4 µm Max-RP C12 column and the chromatograms were acquired at 520, 360, and 320 nm for athocyanins, flavonoids, and free phenolic acids, respectively. The identified anthocyanins were cyanidin-3-glucoside (82.81–91.99% of the total anthocyanins), cyanidin 3,5-di-glucoside (2.31–4.27%), cyanidin-3-rutinoside (1.54–9.20%), peonidin-3-O-glucoside (0.75–3.44%), and pelargonidin-3-glucoside (0.77–2.98%). Other flavonoids (quercetin-3-β-D-glucoside and quercetin-3-rutinoside) and free phenolic acids (chlorogenic and neochlorogenic) were also quantified.     

Metabolite fingerprinting of bokbunja (Rubus coreanus Miquel) by UPLC-qTOF-MS

Metabolite fingerprinting of bokbunja (Rubus coreanus Miquel) using LC-MS according to the cultivation region and the maturity level was performed. The endogenous metabolites were extracted with 80% cold ethanol and the total content of phenolics and anthocyanins compounds was assessed. Accurate mass fingerprinting with chromatographic separation successfully classified bokbunja based on the metabolite profile. Unsupervised classification method (PCA) and supervised prediction model (OPLSDA) provided good capability in categorizing the samples according to the region and the maturity level. Additional tandem-mass spectrometry analysis allowed the identification of some anthocyanin derivatives that showed the most dramatic increase during ripening period (e.g., cyanidin-3-O-glucoside and cyanidin-3-O-rutinoside).     

Simple and fast spectrophotometric determination of low levels of thiabendazole residues in fruit and vegetables after pre-concentration with ionic liquid phase microextraction

There is a great importance of monitoring thiabendazole (TBZ) residues in fruits and vegetables to ensure food safety. Therefore, a new ionic liquid (IL) phase microextraction method using IL, 1-butyl-3-methylimidazoliumhexafluorophosphate [C₄mim][PF₆], as extracting solvent is proposed for simple and fast determination of low levels of TBZ in fruits and vegetables by spectrophotometry. The method is based on selective complex formation of TBZ with Cu(II) ions in presence of PF₆^– as counter ion at pH 5.5, and then microextraction of the complex into the fine micro-drops of IL phase. After optimisation of variables affecting microextraction efficiency, the analytical parameters of the method were determined by calibration curves. The method exhibits a linear relationship (0.3–280 μg L^?1), low detection limit (0.1 μg L^?1), good intra- and inter-day precision (2.4–4.5% as RSD_r%, 2.1–5.6% as RSD_R%), good recovery (≥95.1–98.2%) and high sensitivity enhancement factor (150) by solvent-based calibration curve. It allows a detection limit of 0.24 μg L^?1 and a range of 0.8–250 μg L^?1 by the matrix-matched calibration curve. After validation, the method was successfully applied to the determination of TBZ residues with method quantification limits in fruit and vegetables of 2.0 and 2.5 µg kg^?1 with and without adding polyvinylpyrrolidone (PVP-15) solution. Recoveries range from 85.5% to 98.2% after spiking (10, 50 and 100 µg kg^?1, n: 3).     

Separation,purification of anthocyanin and vitis linn polysaccharide from grape juice by the two‐step extraction and dialysis

Liquid liquid extraction, as a clean and green process, has a great potential in technology and innovation, which has been widely used in industrial production. In this paper, a liquid–liquid extraction process based on L44‐NaH₂PO₄ ATPS was studied for separation and purification of anthocyanin and vitis linn polysaccharide from the grape juice. The concentrations of L44 and NaH₂PO₄, temperature and pH as factors of effect on the extraction rates were discussed. Under the optimal conditions, the extraction rates of anthocyanin and vitis linn polysaccharides were 99.8% and 90.3%, respectively. The work subject is extraction of two compounds from grape juice, which can be very important for food, pharmaceutical and cosmetic industries. The vitis linn polysaccharides were further purified by dialysis and the impurity protein was successfully removed based on organic solvent precipitation method. Besides, the recycle of L44 was also achieved through secondary extraction.

Practical applications

A liquid–liquid extraction process based on L44‐NaH₂PO₄ ATPS was studied for separation and purification of anthocyanin and vitis linn polysaccharide from the grape juice. The efficient separation of two kinds of water‐soluble components is of great significance in industrial production. It is very interesting for scientific communities and shows a great biotechnological potential of the ATPS purification process. The work subject is extraction of two compounds from grape juice, which can be very important for food, pharmaceutical and cosmetic industries.     

Separation of principal component dihydromyricetin from Ampelopsis grossedentata by high-speed counter-current chromatography and its interaction with corn starch

Ampelopsis grossedentata (AG) is an industrial crop in the grape family, which has been used as a dual-purpose plant for medicine and tea with high medicinal values. However, little is reported on the separation technology of active components from AG and processing technology of AG products. High-speed counter-current chromatography (HSCCC) was applied to separate the principal component dihydromyricetin (DMY) from AG. DMY is added to starch-based products to improve food quality. The interaction between corn starch (CS) and DMY was investigated to predict and control the structure and function of starch-based foods. Results show that DMY with 97.13% purity was successfully obtained by HSCCC using a solvent system composed of light petroleum–ethyl acetate–methanol–water–trichloroacetic acid (1:3:1:3:0.01, v/v/v/v/v). Fourier-transform infrared spectroscopy (FT–IR) exhibits that the interactions between CS and DMY included hydrogen bond and noncovalent bond. X-ray diffraction (XRD) shows that DMY could increase the relative crystallinity of CS. Low-field nuclear magnetic resonance results (LF–NMR) imply that DMY decreased the spin relaxation time (T₂) and inhibited the mobility of free water. Atomic force microscopy (AFM) results suggest that DMY changed the surface morphology of CS through hydrogen bond interaction. Moreover, the results of confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM) indicate that DMY could enlarge the pores and change the microstructure of CS–DMY complexes. The findings promote the development of industrial CS-based products and utilization of corn crop.     

Flavonoid glycosides and antioxidant capacity of various blackberry,blueberry and red grape genotypes determined by high‐performance liquid chromatography/mass spectrometry

Anthocyanin and flavonol glycosides of various blackberry, blueberry and red wine grape genotypes were identified and measured by a high‐performance liquid chromatographic (HPLC) separation method with photodiode array (PDA) and mass spectrometric (MS) detection. With this method, two distinct elution regions of anthocyanins and flavonols were obtained with near baseline separation of most compounds. Blackberry, blueberry and red wine grape genotypes varied markedly in total anthocyanins and total flavonols as well as oxygen radical absorbance capacity (ORAC). The respective ranges of total anthocyanin (TA) and total flavonol (TF) contents of tested samples were: blackberries, 1143.9–2415.4 and 102.0–160.2 mg kg^?1; blueberries, 1435.2–8227.3 and 172.5–327.5 mg kg^?1; and red wine grapes, 380.9–7904.7 and 21.0–322.2 mg kg^?1. Antioxidant activities and contents of total anthocyanins and total flavonols in blackberries, blueberries and red wine grapes were highly correlated, with linear relationships between ORAC and TA (r_xy = 0.94) and TF (r_xy = 0.90) for grapes, TA (r_xy = 0.95) for blueberries and TA (r_xy = 0.74) for blackberries. Copyright © 2004 Society of Chemical Industry     

High-intensity ultrasound assisted-emulsification using ionic liquids as novel naturally-derived emulsifiers for food industry applications

Over the last decade, conscious food consumption has been revolutionizing the food industry. The search for additives that meet concepts of naturalness, healthiness and sustainability represents one of the major challenges in food industry. The development of emulsifiers in the ionic liquid (IL) form using compounds that can be obtained from natural sources to replace conventional ones is a promising approach. In this work, we have reported the application of bio-based ILs derived from fatty acids (FAs) and choline (Ch) as novel emulsifiers to produce potential food-grade oil-in-water emulsions by pre-mixing (PM) or PM followed by high-intensity ultrasound (HIUS)-assisted process (PM + U). The effects of the type of IL, considering molecular structures of FAs (C₁₈OOH or C_18:1OOH) and vegetable oil concentration (30:70 or 70:30, oil:water ratio) to form and stabilize emulsions were evaluated. The study was focused on the following aspects: synthesis and characterization of ILs, and kinetic stability, droplet size and size distribution, optical microscopy, viscosity profile and rheological behavior of the emulsions. ILs presented high emulsifier ability, since some emulsions were stable after storage time of at least 30 days, being such stability related to the type of IL, oil concentration and emulsifying process. The observed behavior was associated to lower droplet size and/or emulsion viscosity. [Ch][C₁₈OO] presented higher emulsifier ability than [Ch][C_18:1OO], and this behavior was more evident at higher oil concentration. Emulsions obtained by PM presented phase separation after their preparation, whereas most of those prepared by PM + U were visually stable, being those containing higher oil concentration the formulations that presented higher viscoelasticity, with a gel like behavior. Therefore, bio-based ILs have promising application potential as emulsifiers, and their use to obtain high stable emulsions can be enhanced by using the HIUS technique.     

Extraction and identification of anthocyanin from purple corn (Zea mays L.)

An efficient extraction of anthocyanin from purple corn (Zea mays L.) was investigated in this paper. Tristimulus colourimetry was used to evaluate the process quantitatively and qualitatively. Purple corn anthocyanin was extracted with 1 n HCl–95% ethanol (15:85, v/v) at different extraction temperatures (30–70 °C), times (60–120 min) and solid–liquid ratio (1:20–1:40). The combined effects of extraction conditions on anthocyanin yield and colour attributes were studied using a three‐level three‐factor Box–Behnken design. The results showed that the highest yield of anthocyanin from purple corn (6.02 mg g^?1) were obtained at 70 °C, extraction time 73 min, and solid–liquid ratio 1:25. Three kinds of non‐acylated anthocyanins were detected and characterised as cyanidin‐3‐glucoside, pelargonidin‐3‐glucoside and peonidin‐3‐glucoside by HPLC‐MS.     

Characterisation and preliminary bioactivity determination of Berberis boliviana Lechler fruit anthocyanins

Berberis boliviana Lechler is a member of the Berberidaceae family that has a small edible red-purple berry. The plant is native to the Peruvian Andes and contains high amounts of anthocyanin pigments. The monomeric anthocyanin content, determined by a pH-differential method, was 7/100 g of seedless berries. Pigments were characterised by HPLC coupled to a photodiode array (PDA) and mass spectrophotometer (MS) detectors. Five aglycones and ten anthocyanins were found and identified as petunidin-3-glucoside (24.4%), delphinidin-3-glucoside (24.1%), malvidin-3-glucoside (22.1%), cyanidin-3-glucoside (10.2%), petunidin-3-rutinoside (7.15%), malvidin-3-rutinoside (4.9%), cyanidin-3-rutinoside (3.8%), delphinidin-3-rutinoside (2.6%), peonidin-3-glucoside (1.1%), and peonidin-3-rutinoside (0.9%).     

Determination of Nickel in Food Samples by Flame Atomic Absorption Spectroscopy after Preconcentration and Microextraction Based Ionic Liquids Using Full Factorial and Central Composite Design

Abstract: In this research, a microextraction technique based on ionic liquids (ILs) termed in situ solvent formation microextraction (ISFME) was used for determination of nickel in solutions. 1‐(2‐pyridylazo)‐2‐naphtol (PAN) was chosen as a complexing agent. After preconcentration, the settled IL‐phase was dissolved in 50 μL of ethanol and aspirated into the flame atomic absorption spectrometer (FAAS) using a home‐made microsample introduction system. Injection of 50 μL volumes of analyte into an air–acetylene flame provided very sensitive spike‐like and reproducible signals. ISFME is based on phase separation phenomenon of ionic liquids in aqueous solutions. This method is simple and rapid for extraction and preconcentration of metal ions from food samples and can be applied for the sample solutions containing very high concentrations of salt. Furthermore, this technique is much safer in comparison with the organic solvent extraction because of using ionic liquid. The effective parameters such as amount of IL, salt effect, concentration of the chelating agent and ion pairing agent were inspected by a full factorial design to identify important parameters and their interactions. Next, a central composite design was applied to obtain optimum point of the important parameters. Under the optimum conditions, the calibration graph was linear over the range of 2 to 80 ng/mL. The limit of detection and relative standard deviation (n= 6) were 0.6 ng/mL and 2%, respectively.