• 제목/요약/키워드: HPLC-OPA

검색결과 14건 처리시간 0.026초

추출 용매와 분석 기법에 따른 된장의 유리아미노산 비교 (Comparison of Free Amino Acids in Soybean Paste (Doenjang) by Different Extraction Solvents and Analytical Methods)

  • 강옥주
    • 한국식품조리과학회지
    • /
    • 제23권1호통권97호
    • /
    • pp.150-155
    • /
    • 2007
  • This work was conducted to obtain a rapid, accurate, and precise procedure for free amino acids analysis in Doenjang with HPLC-OPA (high performance liquid chromatography using-phthalaldehyde) and AAA (automatic amino acid analyzer) methods. Different sample extraction procedures among water, 0.1 M perchloric acid, and 0.1% meta-phosphoric acid were also compared. The optimal extraction solvent was 0.1% meta-phosphoric acid for both the HPLC-OPA and AAA methods. Good recoveries for glycine and methionine were observed using the 0.1% meta-phosphoric acid extraction with HPLC-OPA method. Method precisions (% relative standard deviation) for the free amino acids ranged for 1.62% to 8.27%, in which the HPLC-OPA method with water extraction showed the lowest value at 1.62%. Inhibition rates of the free amino acids in Doenjang were greatest with an addition of NaCI at a 1% concentration.

A single-step isolation of useful antioxidant compounds from Ishige okamurae by using centrifugal partition chromatography

  • Kim, Hyung-Ho;Kim, Hyun-Soo;Ko, Ju-Young;Kim, Chul-Young;Lee, Ji-Hyeok;Jeon, You-Jin
    • Fisheries and Aquatic Sciences
    • /
    • 제19권4호
    • /
    • pp.22.1-22.7
    • /
    • 2016
  • One of the main compounds in Ishige okamurae, diphlorethohydroxycarmalol (DPHC), is known to exhibit antiviral and anti-inflammatory effects. However, it has not been investigated extensively. In this study, preparative centrifugal partition chromatography (CPC) coupled with 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) ($ABTS^+$) online HPLC was employed for effectively separating considerable amounts of antioxidant compounds from marine algae. Two main antioxidant compounds, DPHC and octaphlorethol A (OPA), respectively, were confirmed and isolated from the ethyl acetate (EtOAc) fraction of I. okamurae by $ABTS^+$ online HPLC and preparative CPC systems. The presence of DPHC and OPA was confirmed in the EtOAc fraction of I. okamurae by both liquid chromatography with diode array detection and electrospray ionization mass spectrometry (LC-DAD-ESI/MS) and $ABTS^+$ online HPLC systems: DPHC (39 mg) and OPA (23 mg) were successfully isolated from I. okamurae (500 mg) with optimum solvent composition (0.5:10:4:6; n-hexane/EtOAc/MeOH/water, v/v) with corresponding partition coefficients (K) of 1.62 and 2.71, respectively, by preparative CPC. Hence, CPC coupled with $ABTS^+$ online HPLC is convenient for the efficient and simple isolation of these antioxidant compounds from I. okamurae.

Improved Fluorescent Determination Method of Cellular Sphingoid Bases in High-performance Liquid Chromatography

  • Yoon, Hong-Tak;Yoo, Hwan-Soo;Shin, Bum-Kyu;Lee, Woo-Jin;Kim, Hwan-Mook;Hong, Seon-Pyo;Moon, Dong-Cheul;Lee, Yong-Moon
    • Archives of Pharmacal Research
    • /
    • 제22권3호
    • /
    • pp.294-299
    • /
    • 1999
  • Precolumn orthophthaldehyde (OPA) labeling method of sphingoid bases, sphingosine and sphinganine, was investigated to obtain high fluorescent detectability. In order to improve the fluorescent yield, we investigated the optimal solubility of sphingoid bases for five pre-incubation solvents by incorporating the heating procedure before OPA derivatization. The pre-incubation in ethanol prominently increased the fluorescent peak height of OPA derivative for each sphingoid bases in high performance liquid chromatography. About tenfold increase of detectability was archived by pre-incubating lipid extracts pellets in ethanol at $60^{\circ}C$ for 30 min. Optimal derivatization was performed in 30 min at ambient temperature and the fluorescent intensity of OPA derivative was stable for two weeks at $4^{\circ}C$. The detection limit of sphingosine was 0.1 pmol as injected amount. This method was applied to the determination of cellular sphingosine and sphinganine in various human lung cancer cells. This OPA procedure was prospective to be useful for quantitating the amount of sphingoid bases in other cancer cells.

  • PDF

HPLC-ECD를 이용한 흰쥐의 뇌 중 감마 아미노부티르산 및 글루탐산의 정량 (Determination of ${\gamma}-Aminobutyric$ Acid and Glutamic Acid in Rat Brain by High Performance Liquid Chromatography with Electrochemical Detection)

  • 강종성;이순철
    • 약학회지
    • /
    • 제43권3호
    • /
    • pp.300-305
    • /
    • 1999
  • A sensitive and efficient assay method was applied to determine the level of glutamic acid (GA) and ${\gamma}-aminobutyric$ acid (GABA) in frontal cortex and hippocampus of rat administrated with ethanol and drugs. The compounds were derivatized with ο-phthalaldehyde (OPA) and 2-mercaptoethanof for precolunm analysis. The condition for the simultaneous determination of GA, GABA and $\beta-aminobutyric$ acid (BABA) by high performance liquid chromatography with electrochemical detection was reverse phase $C_{18}$ column as stationary phase, 0.1 M phosphate buffer containing 0.1 mM $Na_4EDTA$ : methanol = 55:45 (v+v) pH 3.8 as mobile phase and 0.7V electrode voltage. The stability of reaction product of GA, GABA and BABA with OPA could be increased by adding the same volume of polyethylene glycol 400 to reaction mixture. The GABA level in frotal cortex of rat was significantly decreased by the administration of picrotoxin and diazepam, but it was significantly increased by the administration of red ginseng total saponin, N-methyl-D-glucamine and (-)-deprenyl.

  • PDF

생체시료 중 미량 아미노산 대사 프로필을 위한 분석법 응용 (Applied Analysis for Metabolic Profiling of Trace-level Amino Acid in Biological Fluid)

  • 남형욱;박송자;표희수;팽기정
    • 분석과학
    • /
    • 제16권5호
    • /
    • pp.349-357
    • /
    • 2003
  • 유전학적으로 밝혀지지 않은 생물학적 연구에 있어서 작은 분자량의 아미노산 또는 홀몬과 같은 대사체들은 그 변화와 여러 생물학적 데이타와 결합 되어 직접적인 생화학적 의미 해석을 가능하게 한다. 미량의 생체 시료에 존재하는 아미노산을 분석하기 위해서 HPLC/FLD를 사용하였으며, 감도가 우수하고 반응시간이 빠른 유도체화 방법인 OPA/3-MPA로 형광 유도체화 하여 세포 배지를 바탕시료로 하여 아미노산을 분석하였다. 유도체화물의 시간에 대한 불안정성을 개선하기 위하여 다 단계의 injector program을 사용하여 유도체화 반응 후 시료 주입시간을 일정하게 조절하여 유도체화 과정 중 발생할 수 있는 불순물 제거 및 정량성을 개선하였다. 19종 아미노산의 표준 검정 곡선은 0.5 - 100.0 ppb의 범위에서 $r^2=0.99$ 이상의 직선성을 나타냈으며, 검출 한계는 1.70 pmol(GLU) - 23.81 pmol(SER) 범위로 측정되었다. 이를 통해 다량의 세포를 대상으로 하는 대사 프로필을 위해 감도가 우수하고 안정적으로 정량할 수 있는 분석법을 설정하였다.

Analysis of intracellular amino acids in the fermentation of Pichia pastoris X-33 and KM71H

  • Han, Kyung-Ah;Kim, Sun-Yong;Rhee, Jong-Il
    • 한국생물공학회:학술대회논문집
    • /
    • 한국생물공학회 2005년도 생물공학의 동향(XVII)
    • /
    • pp.651-654
    • /
    • 2005
  • 10 g/L yeast extract, 20 g/L peptone, 20 g/L dextrose가 첨가된 YPD 배지를 이용하여 200 rpm, 30 $^{\circ}C$에서 24시간 배양한 Pichia pastoris X-33과 KM71H를 각각 대수 증식기에 시료를 채취하여 4000 g, 4 $^{\circ}C$에서 5분간 원심분리하여 펠릿만을 얻어 멸균된 증류수를 500 ${\mu}{\ell}$ 넣고 4000 g, 4 $^{\circ}C$에서 5분간 원심분리하여 2-3회 세포 내 단백질과 아미노산 분석에 이용하였다. HPLC는 OPA 시약을 만들어 4 $^{\circ}C$에서 24시간 안정화시켜 사용하였고, 용액 A와 B를 미리 준비하여 기울기용리로 30 $^{\circ}C$에서 1ml/min, Ex 330/ Em 420 nm에서 80분간 분석하였다.

  • PDF

건강기능식품에서 HPLC를 이용한 4-Hydroxy-L-isoleucine 분석법 연구 (Development of Method for Determining 4-Hydroxy-L-isoleucine in Health Functional Foods by HPLC)

  • 이세윤;정희선;허수정;이혜영;오재명
    • 한국식품위생안전성학회지
    • /
    • 제35권3호
    • /
    • pp.213-218
    • /
    • 2020
  • 본 연구는 건강기능식품 호로파종자식이섬유 중 4-hydroxyL-isoleucine에 대한 분석법을 개발하는 연구이다. 최적분석 조건을 확립하기 위해 시료 채취량, 전처리 용매 및 이동상 용매 조건을 비교 검토하였으며, HPLC-PDA를 이용하여 시료 중의 4-hydroxy-L-isoleucine를 분석하였다. 분석 시 사용한 컬럼은 Capcell Pack UG120 C18 (Shiseido, 4.6×250 mm, 5 ㎛)이며, 유도체 시약으로 OPA를 선정하였다. 확립된 시험법에 대해 특이성, 직선성, 검출한계, 정량한계, 정확성, 정밀성 등의 밸리데이션을 수행하였다. 5-100 ㎍/mL 농도에서 결정계수(R2) 0.999 이상으로 높은 직선성을 확인하였다. 또한, 회수율은 91.7-96.4%이었고, 정밀성은 0.5-1.1%의 상대표준편차(%RSD)를 확인하였다. 개발된 시험법은 호로파종자추출물 중 4-hydroxy-L-isoleucine 분석을 위한 시험법으로 활용되기에 적합한 것으로 판단된다.

Stability Test and Quantitative and Qualitative Analyses of the Amino Acids in Pharmacopuncture Extracted from Scolopendra subspinipes mutilans

  • Cho, GyeYoon;Han, KyuChul;Yoon, JinYoung
    • 대한약침학회지
    • /
    • 제18권1호
    • /
    • pp.44-55
    • /
    • 2015
  • Objectives: Scolopendra subspinipes mutilans (S. subspinipes mutilans) is known as a traditional medicine and includes various amino acids, peptides and proteins. The amino acids in the pharmacopuncture extracted from S. subspinipes mutilans by using derivatization methods were analyzed quantitatively and qualitatively by using high performance liquid chromatography (HPLC) over a 12 month period to confirm its stability. Methods: Amino acids of pharmacopuncture extracted from S. subspinipes mutilans were derived by using O-phthaldialdehyde (OPA) & 9-fluorenyl methoxy carbonyl chloride (FMOC) reagent and were analyzed using HPLC. The amino acids were detected by using a diode array detector (DAD) and a fluorescence detector (FLD) to compare a mixed amino acid standard (STD) to the pharmacopuncture from centipedes. The stability tests on the pharmacopuncture from centipedes were done using HPLC for three conditions: a room temperature test chamber, an acceleration test chamber, and a cold test chamber. Results: The pharmacopuncture from centipedes was prepared by using the method of the Korean Pharmacopuncture Institute (KPI) and through quantitative analyses was shown to contain 9 amino acids of the 16 amino acids in the mixed amino acid STD. The amounts of the amino acids in the pharmacopuncture from centipedes were 34.37 ppm of aspartate, 123.72 ppm of arginine, 170.63 ppm of alanine, 59.55 ppm of leucine and 57 ppm of lysine. The relative standard deviation (RSD %) results for the pharmacopuncture from centipedes had a maximum value of 14.95% and minimum value of 1.795% on the room temperature test chamber, the acceleration test chamber and the cold test chamber stability tests. Conclusion: Stability tests on and quantitative and qualitative analyses of the amino acids in the pharmacopuncture extracted from centipedes by using derivatization methods were performed by using HPLC. Through research, we hope to determine the relationship between time and the concentrations of the amino acids in the pharmacopuncture extracted from centipedes.

N-oleoyl-D-erythro-sphingosine-based Analysis of Ceramide by High Performance Liquid Chromatography and Its Application to Determination in Diverse Biological Samples

  • Lee, Youn-Sun;Choi, Heon-Kyo;Yoo, Jae-Myung;Choi, Kyong-Mi;Lee, Yong-Moon;Oh, Sei-Kwan;Kim, Tack-Joong;Yun, Yeo-Pyo;Hong, Jin-Tae;Okino, Nozomu;Ito, Makoto;Yoo, Hwan-Soo
    • Molecular & Cellular Toxicology
    • /
    • 제3권4호
    • /
    • pp.273-281
    • /
    • 2007
  • Ceramide is involved in cell death as a lipid mediator of stress responses. In this study, we developed an improved method of ceramide quantification based on added synthetic ceramide and thin layer chromatography (TLC) separation, and applied to biological samples. Lipids were extracted from samples spiked with N-oleoyl-D-erythro-sphingosine ($C_{17}$ ceramide) as an internal standard. Ceramide was resolved by TLC, complexed with fatty-acidfree bovine serum albumin (BSA), and deacylated by ceramidase (CDase). The released sphingosine was derivatized with o-phthalaldehyde (OPA) and measured by high performance liquid chromatography (HPLC). The limit of detection for ceramide was about 1-2 pmol and the lower limit of quantification was 5 pmol. Ceramide recovery was approximately 86-93%. Ceramide concentrations were determined in biological samples including cultured cells, mouse tissues, and mouse and human plasma. TLC separation of ceramide provides HPLC chromatogram with a clean background without any interfering peaks and the enhanced solubility of ceramide by BSAceramide complex leads to the increased deacylation of ceramide. The use of an internal standard for the determination of ceramide concentration in these samples provides an accurate and reproducible analytical method, and this method can be applicable to diverse biological samples.

Xylazine이 histamine 유리에 미치는 영향 (Effect of xylazine hydrochloride on histamine release)

  • 김영환;박준형
    • 한국동물위생학회지
    • /
    • 제25권1호
    • /
    • pp.53-73
    • /
    • 2002
  • It has been reported that degranulation of mast cells in rats, rabbits and dog was observed after dosing xylazine hydrochloride(Xh) which has been widely used as sedative, analgesic and muscular relaxant. Therefore, this experiment was conducted to examine the relations between Xh and histamine release and to identify the action of ${\alpha}$-adrenoceptors which exists on the suface of mast cells. 1. The content of histamine within serum was measured with HPLC by performing the O-phthalaldehyde(OPA) fluorescent derivation. The pretreatment method had a little modification from the conventional method. The pretreament was carried out in the following method. 0.2$m\ell$ of serum and 1$m\ell$ of butanol were added to mixed together and then the liquid was centrifugally separated at 4$^{\circ}C$ and 2,000 rpm for 3 minutes. 0.4$m\ell$ of 0.1N HCl and 1.6$m\ell$ of heptane were added to 0.8$m\ell$ of supernatant taken from the liquid, and they were mixed together. This mixture was also centrifugally separated at 4$^{\circ}C$ and 2,000 rpm for 5 minutes. The supernatant was thrown away and the OPA fluorescent derivation was carried out with 0.2$m\ell$ of the lower liquid then, 5 minutes after mixing 400${\mu}\ell$ of 0.1N HCl, 120${\mu}\ell$ of 1N NaOH and 40${\mu}\ell$ of 0.1% OPA in the 0.2$m\ell$ of the lower liquid,120${\mu}\ell$ of 3.57N H$_3$PO$_4$ was added to the mixed liquid, and the liquid, was mixed again and syringe-filtered. Then, the measurement was done with HPLC in the 30 : 70(ν/ν) ratio of 0.004M KH$_2$PO$_4$: CH$_3$CN, flow rate of 1.0$m\ell$/min., and a wavelength of λex= 350nm and λem=444nm at the column temperature of 27$^{\circ}C$, using the fluorescence detector. 2. The content of histamine in each laboratory animal appeared to be higher in such an order as rabbit, rat, guinea pig, dog, Korean indigenous goat, swine, Korean indigenous cattle, Holstein, and mouse, of which the individual mean values${\pm}$standard deviation were 2.0668 ${\pm}$ 0.6049. 0.4999 ${\pm}$ 0.2278, 0.4241 ${\pm}$ 0.1974, 0.1054 ${\pm}$ 0.0556, 0.1028 ${\pm}$ 0.0276, 0.0972 ${\pm}$ 0.0513, 0.0872 ${\pm}$ 0.0373, 0.0717 ${\pm}$ 0.0379, and 0.0706 ${\pm}$ 0.0366, respectively. 3. The content of histamine was measured at the moments of 15-, 30-, 60-, 120-minutes after inoamuscular injection of 20mg/100kg Xh into two to 4 years old Holstein weighing 600∼700kg. The result showed that there was a significant increase at the times of 30- and 90-minutes after injection(p<0.05). 4. Intramuscular injection of 3mg/10kg Xh was given to crossbred pug dogs weighing 2.5∼4.3kg. The content of histamine was measured at the times of 30-, 60-, 90- and 120-minutes after injection. The result revealed that there was a significant increase at the times of 60-and 90-minutes after injection(p<0.05). 5. Intramuscular injection of 10mg/$m\ell$∼25mg/$m\ell$ Xh in concentration of 0.1$m\ell$ was applied to Korean indigenous goat over 5 months old. Then, the content of histamine was measured at the times of 15-, 30-, 60- and 90-minutes after injection. A significant increase was shown at the times of 30- and 60-minutes after injection(p<0.05). 6. The content of histamine was measured at the moments of 30- and 60-minutes after intramuscular injection of 0.1-0.2$m\ell$ Xh (20mg/$m\ell$) into male rabbits weighting 2.5-4kg. A significant increase was found at the moment of 60 minutes after injection(p<0.001). 7. After administering Xh to the mast cell taken from the abdominal cavity of mouse, the content of histamine was measured. The result showed that the higher the concentration, the more significantly the content of histamine was increased(p<0.05). 8. Compound 48/80 was administered in concentration of 5$\mu\textrm{g}$/$m\ell$ and 10$\mu\textrm{g}$/$m\ell$ to the mast cell picked from the abdominal cavity of mouse. The result showed that there was a significant increase in the content of histamine in case of the concentration of 10$\mu\textrm{g}$/$m\ell$(p<0.05). It was found to be about 10,000 to 500,000 times stronger than the Xh. 9. After premedication of 1mg/kg of yohimbine hydrochloride as ${\alpha}$$_2$-adrenergic antagonist to rabbits, the Xh was administered to them. The result was that the value of histamine within serum was decreased significantly(p<0.001). 10. After premeditation of 1mg/kg of prazosin hydrochloride as ${\alpha}$$_1$-adrenergic antagonist to rabbits, the Xh was administered to them. It was found that the value of histamine within serum was decreased significantly(p<0.005). 11, Prazosin hydrochloride and yohimbine hydrochloride as ${\alpha}$$_1$-adrenergic antagonist, respectively, and ${\alpha}$$_2$-adrenergic antagonist were administerd. In this case, the value of histamine within serum was decreased significantly(p<0.0001). As the results, when the Xh is administered to various kinds of animals, the amount of histamine release within serum is increased. In view of the results so far achieved, it is concluded that Xh acted on both a$_1$-adrenoreceptor and ${\alpha}$$_2$-adrenoreceptor induces the degranulation of mast cell.