• Title/Summary/Keyword: Elution development

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

  • Lee, Se-Yun;Jeong, Hee-Sun;Hu, Soo-Jung;Lee, Hye-Young;Oh, Jae-Myoung
    • Journal of Food Hygiene and Safety
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    • v.35 no.3
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    • pp.213-218
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    • 2020
  • This study was conducted to develop a method of analysis for 4-hydroxy-L-isoleucine in the seed extract of fenugreek (Trigonella foenum graecum), a health functional food that contains dietary fiber. The analytical method for 4-hydroxy-L-isoleucine was derived with O-phthaldialdehyde reagent (OPA) and determined by HPLC-PDA. The method was performed on a Capcell Pak C18 UG 120 column (4.6×250 mm, 5 ㎛) in isocratic elution mode using disodium phosphate and acetonitrile. The validation of the developed analytical method was conducted by evaluating several parameters; selectivity, linearity, limit of detection (LOD), limit of quantification (LOQ), accuracy and repeatability. Excellent linearity (R2=0.999) was observed for 4-hydroxy-L-isoleucine in the concentration range (5-100 ㎍/mL). Observed recoveries of these compounds were found to be between 91.7 and 96.4%. Precision was between 0.2 and 2.4% relative standard deviation (%RSD).

Simultaneous Determination of Five Porphyrins in Human Urine and Plasma Using High Performance Liquid Chromatography-Tandem Mass Spectrometry

  • Hur, Yeoun;Tae, Sookil;Koh, Yun-Joo;Hong, Sung-Hyun;Yoon, Young Ho;Jang, Haejong;Kim, Sooji;Kim, Kyeong Ho;Kang, Seung Woo;Lee, Youngshin;Han, Sang Beom
    • Mass Spectrometry Letters
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    • v.5 no.2
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    • pp.42-48
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    • 2014
  • A specific and sensitive liquid chromatography-electrospray ionization tandem mass spectrometry method (LC-ESI-MS/MS) was developed and validated for the simultaneous quantification of porphyrins (coproporphyrin, pentacarboxylporphyrin, hexacarboxylporphyrin, heptacarboxylporphyrin, and uroporphyrin) in human plasma and urine. Acidified plasma samples and urine samples were prepared by using liquid-liquid extraction using ethyl acetate and protein precipitation with acetonitrile, respectively. The separation was achieved onto a Synergi Fusion RP column ($150mm{\times}2.0mm$, $4{\mu}m$) with a gradient elution of mobile phase A (0.1% formic acid in 2 mmol/L ammonium acetate, v/v) and mobile phase B (20% methanol in acetonitrile, v/v) at a flow rate of $450{\mu}L$/min. Porphyrins and the internal standard (IS), coproporphyrin I-$^{15}N_4$, were detected by a tandem mass spectrometer equipped with an electrospray ion source operating in positive ion mode. Multiple reaction monitoring (MRM) transitions of the protonated precursor ions and the related product ions were optimized to increase selectivity and sensitivity. The proposed method was validated by assessing selectivity, linearity, limit of quantification (LOQ), precision, accuracy, recovery, and stability. The calibration curves were obtained in the range of 0.1-100 nmol/L and the LOQs were estimated as 0.1 nmol/L for all porphyrins. Results obtained from the validation study of porphyrins showed good accuracy, precision, recovery, and stability. Finally, the proposed method was successfully applied to clinical studies on the autism spectrum disorder (ASD) diagnosis of 203 Korean children.

Overexpression and Periplasmic Transport of 5-Enolpyruvylshikimate 3-Phosphate Synthase in E. coli (대장균에서 5-Enolpyruvylshikimate 3-Phosphate Synthase의 대량 발현 및 Periplasmic Space로의 Transport)

  • 김남일;임재윤;조태주
    • Korean Journal of Microbiology
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    • v.33 no.1
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    • pp.1-6
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    • 1997
  • 5-Enolpyruvylshikimate 3-phosphate(EPSP) synthase is the sixth enzyme of the shikimate pathway that synthesizes aromatic amino acids. The enzyme is a primary target for the glyphos'lte which is a broad-spectrum and environmetally safe herbicide. As a first step toward development of glyphpsate-resistant EPSP synthase, the EPSP synthase gene(aroA) was amplified by polymerase chain reaction and cluned into pET-25b vector. In this construct. designated pET-aro, the aroA gene is expressed under control of strong T7 promoter. and the EPSP synthase is produced as a fusion protein with pelB leader at N-terminus and HSV-tag and His-tag at C-terminus. When the pET-aro clone was induced to produce the enzyme, it was found that the EPSP synthase was successfully exported to peri plasmic space. The periplasmic transport was greatly dependent on the induction temperatures. Among the induction temperatures examined($25^{\circ}C$, $30^{\circ}C$, $34^{\circ}C$ and $37^{\circ}C$). induction at $34^{\circ}C$ gave rise to maximal periplasmic transport. The recomhinant EPSP synthase could have been purified hy $Ni^{2+}$ -affinity chromatography using the His-tag. and detected hy anti-HSV -tag antibody. The recombinant EPSP synthase also hound to phosphocellulose resin and was eluted hy shikimate 3-phosphate and phosphoenolpyruvate. as expected. The recombinant EPSP synthase purified from phosphocellulose resin showed typical EPSP synthase activity.

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Isolation and Development of Quantification Method for Cyanidin-3-Glucoside and Cyanidin-3-Rutinoside in Mulberry Fruit by High-Performance Countercurrent Chromatography and High-Performance Liquid Chromatography

  • Choi, Soo-Jung;Jeon, Heejin;Lee, Chang Uk;Yoon, Shin Hee;Bae, Soo Kyung;Chin, Young-Won;Yoon, Kee Dong
    • Natural Product Sciences
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    • v.21 no.1
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    • pp.20-24
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    • 2015
  • Cyanidin-3-glucoside (C3G) and cyanidin-3-rutinoside (C3R) were isolated by high-performance countercurrent chromatography (HPCCC) using a two-phase solvent system composed of tert-butyl methyl ether/n-butanol/acetonitrile/water/trifluoroacetic acid (1 : 3 : 1 : 5 : 0.01, v/v) to give pure C3G (34.1 mg) and C3R (14.3 mg) from 1.5 g crude mulberry fruit extract. Using the pure C3G and C3R, a reliable high-performance liquid chromatography (HPLC) method was developed and validated to determine the C3G and C3R contents in mulberry fruit. C3G and C3R were separated simultaneously using an Eclipse XDB-C18 column ($4.6{\times}250mm$ I.D., $5{\mu}m$) coupled with a photodiode array detector (PDA). The gradient elution of the mobile phase consisting of acetonitrile (0.5% formic acid) and water (0.5% formic acid) was applied (1.0 mL/min), and the detection wavelength was 520 nm. The calibration curves of C3G and C3R showed good linearity (both with $r^2=0.9996$) in the concentration range $15.625-500{\mu}g/mL$, and the relative standard deviations (RSD%) of intra- and inter-day variability were in the ranges 2.1 - 8.2% and 4.1 - 17.1%, respectively. The accuracies were ranged 96.5 - 102.6% for C3G and C3R, respectively. The developed HPLC method was used to determine the contents of C3G and C3R in newly harvested mulberry from eight different provinces of Korea.

Characterization of tissue conditioner containing chitosan-doped silver nanoparticles (키토산-은나노 복합체가 함유된 의치 연성이장재 특성에 관한 연구)

  • Nam, Ki Young;Lee, Chul Jae
    • The Journal of Korean Academy of Prosthodontics
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    • v.58 no.4
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    • pp.275-281
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    • 2020
  • Purpose: Development of a latent antimicrobial soft liner is strongly needed to overcome a possible inflammation related with its dimensional degrade or surface roughness. Modified tissue conditioner (TC) containing chitosan-doped silver nanoparticles (ChSN) complexes were synthesized and assessed for their characterizations. Materials and methods: ChSN were preliminarily synthesized from silver nitrate (AgNO3), sodium borohydride (NaBH4) as a reducing agent and chitosan biopolymer as a capping agent. Ultraviolet-visible and Fourier transform infrared spectroscopy were conducted to confirm the stable reduction of nanoparticles with chitosan. Modified TC blended with ChSN by 0 (control), 1.0, 3.0 and 5.0 % mass fraction were mechanically tested by ultimate tensile strength (UTS), silver ion elution and color stability (n=7). Results: At 24 hour and 7 day storage periods, UTS values were not significant (P>.05) as compared with pristine TC (control) and silver ion was detected with the dose-dependent values of ChSN incorporated. Color stability of TC were influenced by ChSN add, with the higher doses, the significantly greater color changes (P<.05). Conclusion: A stable synthesized ChSN was acquired and modified TC loading ChSN was characterized as silver ion releasing without detrimental physical property. For its clinical application, antimicrobial test, color control and multifactor investigations are still required.

Applicability of SRSL(Self-Recovering Sustainable Liner) to the Landfill Final Cover System (SRSL(Self Recovering Sustainable Liner)재의 매립지 최종복토층에 대한 활용성 검토)

  • Kwon, Oh-Jung;Seo, Min-Woo;Hong, Soo-Jung;Park, Jun-Boum;Park, Soo-Young
    • Proceedings of the Korean Geotechical Society Conference
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    • 2004.03b
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    • pp.453-460
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    • 2004
  • To prevent penetration of rainwater into the landfill site is the main purpose of the final cover in landfill sites. Conventional designs of landfill covers uses geotextiles such as geomembrane and GCL, and clay liners to lower the permeability of final covers of landfill sites. However, differential settlement and the variation of temperature in landfill sites cause the development of cracks or structural damage inside the final cover and it is also difficult to obtain clay - the main material of the compacted clay liner in Korea. Thus the former final cover system that suggests geomembrane and GCL or compacted clay liner has several limitations. Therefore, an alternative method is necessary and one of them is the application of SRSL(self-Recovering Sustainable Liner) material. SRSL is two different layers consist of individual materials that react with each other and form precipitates, and with this process lowers the permeability of the landfill final cover. SRSL generally is made up of two layers, so that when a internal crack occurs the reactants of the two layers migrate towards the crack and heal it by forming another liner. In this study the applicability of SRSL material for landfill final cover was examined by performing; (1) jar test to verify the formation of precipitate in the mixture of each reactants, (2) falling head test considering the field stress in order to confirm the decrease of permeability or prove that the hydraulic condctivity is lower than the regulations, (3) compression tests to judge weather if the strength satisfies the restricts for landfills, (4) freeze/thaw test to check the applicability of SRSL for domestic climate. In addition, the application of waste materials in the environmental and economical aspect was inspected, and finally the possibility of secondary contamination due to the waste materials was examined by performing elution tests.

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The Endotoxin Assay of Contaminated Titanium Implants following Various Techniques of Detoxification (오염된 임프란트 표면의 해독 방법에 따른 내독소 제거 효과에 관한 연구)

  • Park, Joong-Hee;Lim, Sung-Bin
    • Journal of Periodontal and Implant Science
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    • v.34 no.1
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    • pp.71-81
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    • 2004
  • Peri-implantitis could be the result of biomechanical and occlusal overload as well as microbiologic invasion. The dental implant may be more susceptible to dental plaque than the natural tooth, as the predictability of a stable soft tissue attachment complex has not yet been confirmed. With the development of peri-implantitis, the implant surface would be exposed to the oral environment and becomes coated with bacteria. The objective of therapy for this condition is to regain integration of the implant with bone. Since fibroblast adherence to surfaces is impeded by endotoxin, it would seem that decontamination would be desirable to obtain maximum osseointegration. The purpose of this study was to determine whether various chemotherapeutic and mechanical treatments(distilled water, air-powder abrasive, hypersaturated citric acid, tetracycline) can detoxify contaminated titanium implant surface by means of kinetic LAL test. Experimental rough surface titanium disks were fabricated. All of them were divided into two groups(A.a group and P.g group) and each contaminated by A. actinomycetemcomitans and P. gingivalis suspension. Contaminated disks were treated with distilled water, air-powder abrasive, citric acid and tetracycline, and then all disks were placed into LPS-free water for elution. The results were as follows : 1. In A.a group, LPS elute level of all test groups were significantly lower than control group(p<0.05). 2. In A.a group, LPS elute level of test 2, test 3 and test4 groups were significantly lower than that of control group(p<0.05). But, among the test 2, test 3, test4 groups, the significant differences were not detected. 3. In P.g group, LPS elute level of test 2, test 3 and test 4 groups were lower than that of control group(p<0.05). But, among the test groups, the significant differences were not detected. From the result of this study, it would be concluded that air-powder abrasive, hypersaturated citric acid and tetracycline treatments may be effective at reducing endotoxin level on rough titanium implant surfaces, and can be clinically used. But the treatments in peri-implantitis differentially impact osseointegration making one method clinically superior. To gain this knowledges, further molecular biologic and histopathologic studies should be developed.

Development of Method for Analysis of Four Sulfonylurea Pesticides, Rimsulfuron, Ethametsulfuron-methyl, Tribenuron-methyl, Chlorimuron-ethyl Residues by High-Performance Liquid Chromatography with Diode-Array Detection (HPLC/DAD를 이용한 림술푸론, 에타메트설푸론메틸, 트리베누론메칠, 클로리무론에칠 4종 성분의 잔류농약 분석법 개발)

  • Koo, Yun-Chang;Yang, Sung-Yong;Wang, Zeng;An, Eun-Mi;Heo, Kyoung;Kim, Hyeng-Kook;Shin, Han-Seung;Lee, Jin-Won;Lee, Kwang-Won
    • Journal of the Korean Society of Food Science and Nutrition
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    • v.39 no.8
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    • pp.1231-1235
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    • 2010
  • The method for residue analysis of four sulfonylurea pesticides, rimsulfuron, ethametsulfuron-methyl, tribenuron-methyl and chlorimuron-ethyl was examined and analyzed by HPLC with ODS column ($250\;mm{\times}4.6\;mm$, $5\;{\mu}m$ diameter particle size) which was maintained at $35^{\circ}C$. Mobile phase consisted of solvent A (20 mM $KH_2PO_4$, pH 2.5) and solvent B (acetonitrile). Isocratic elution of the column with 45% solvent A and 55% solvent B at a flow rate of 1 mL/min resulted in retention times of 5.92, 6.54, 9.28, and 14.35 min for rimsulfuron, ethametsulfuron-methyl, tribenuron-methyl, and chlorimuron-ethyl, respectively. All injection volumes were $20\;{\mu}L$. The limit of quantitation was 0.02, 0.01, 0.001, and 0.004 mg/kg for rimsulfuron, ethametsulfuron-methyl, tribenuron-methyl, and chlorimuron-ethyl, respectively. Recovery rate test was performed with three farm products, rice, apple and soybean. Four sulfonylurea pesticides were spiked at concentrations of 0.05, 0.1 and 0.5 mg/kg. The recovery rates were ranged from 86.12% to 116.26% and the standard deviations of all experiments were within 10%.

Development of an Analytical Method for the Determination of Dexamethasone in Bovine Milk Using Liquid Chromatography Coupled to Tandem Mass Spectrometry (LC-MS/MS를 이용한 우유 중 덱사메타손의 잔류 분석법 개발)

  • Cha, Chun-Nam;Park, Eun-Kee;Yoo, Chang-Yeul;Lee, Sung Joong;Son, Song-Ee;Kim, Suk;Lee, Hu-Jang
    • Journal of Food Hygiene and Safety
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    • v.32 no.5
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    • pp.418-423
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    • 2017
  • An analytical method for the determination of dexamethasone (DM) in bovine milk samples was developed and validated using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Milk samples were extracted by the liquid-liquid extraction based on acetonitrile. The chromatographic separation was achieved on a reverse phase $C_{18}$ column with gradient elution using a mobile phase of 0.1% formic acid in 95% acetonitrile. The procedure was validated according to the Ministry of Food and Drug Safety guideline determining accuracy, precision, limit of detection (LOD), and limit of quantification (LOQ). Mean recoveries of DM from spiked milk samples (25, 125, and 1,250 ng/mL) were 98.9-109.6%, and the relative standard deviation was between 1.7 and 4.4%. Linearity in concentration range of 12.5-1,250 ng/mL was obtained with the correlation coefficient ($r^2$) of 0.9997. LOD and LOQ for the investigated DM were 0.15 and 0.5 ng/mL depending on milk samples, respectively. This method was reliable, sensitive, economical and suitable for routine monitoring of DM residues in bovine milk.

Development of Separation and Trace Analysis Methods for Platinum Group Elements-Separation and Retention Behavior of Platinoid Metal Acetylacetonates in Reversed-Phase Liquid Chromatography (백금족 원소의 분리 및 미량분석법 개발에 관한 연구: 역상 액체 크로마토그래피에 의한 백금족 금속-아세틸아세톤 킬레이트들의 분리 및 머무름 거동)

  • Lee, Dai Woon;Kim, Kyung Soo;Park, Young Hun;Czea, Myoung Zoon;Chung, Koo Soon
    • Analytical Science and Technology
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    • v.6 no.1
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    • pp.107-119
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    • 1993
  • The purpose of this study is to investigated the elution behavior of platinoid metal acetylacetonates, which is the key to elucidate their retention mechanism and optimize their RPLC separation conditions. The retention data of four platinoid metal acetylacetonates have been measured on four different columns in methanol-water and acetonitrile-water systems. The retention of uncharged platinoid metal acetylacetonates is interpreted by solvophobic effect. The retention of platinoid metal acetylacetonates is also greatly influenced by the geometric structure of the complexes. The square planar chelates, $Pd(acac)_2$, $Pt(acac)_2$, are retained longer than the octahedral chelates, $Rh(acac)_3$, $Ir(acac)_3$. It is likely due to that square planar chelates show greater interaction with nonpolar stationary phase than octahedral chelates. The results of van't Hoff plots have shown that platinoid metal acetylacetonates is operated on the same retention mechanism in the temperature range of $25{\sim}45^{\circ}C$. The study of the retention mechanism by the enthalpy-entropy compensation phenomenon has indicated that the retention mechanism of octahedral chelates and square planar chelates do not vary with the composition change of methanol-water mobile phase, respectively. In acetonitrile-water mobile phase, however, the retention mechanism is observed to be more complicated. Optimum condition for the separation of four platinoid metal acetylacetonates is found to be 40% methanol, polymeric C18 column, and $45^{\circ}C$.

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