• Title/Summary/Keyword: high performance liquid chromatography

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Production and Characterization of a Monoclonal Antibody Against Enrofloxacin

  • Chusri, Manaspong;Wongphanit, Pitikarn;Palaga, Tanapat;Puthong, Songchan;Sooksai, Sarintip;Komolpis, Kittinan
    • Journal of Microbiology and Biotechnology
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    • v.23 no.1
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    • pp.69-75
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    • 2013
  • Enrofloxacin is a fluoroquinolone antibiotic approved for the treatment of infections in animals. Because of the side effects to consumers of animal products, the maximum residue limits (MRLs) of enrofloxacin in animal tissues for consumption are regulated. In this study, a monoclonal antibody (mAb) against enrofloxacin was prepared and characterized for the development of a direct competitive enzyme-linked immunosorbent assay (ELISA). The obtained mAb, Enro44, was highly specific for enrofloxacin and had a 50% inhibition concentration ($IC_{50}$) of 1.99 ng/ml in a competitive ELISA, and the limit of detection (LOD) was 0.50 ng/ml. The cross-reactivity of the mAb with other quinolones and fluoroquinolones was lower than 0.01%. The subclass of the mAb Enro44 was identified as IgG1. The antigen (Ag)-captured direct competitive ELISA using the mAb Enro44 was tested on different spiked samples, including chicken muscle, cattle milk, and cattle urine, and the assay demonstrated recoveries of 82-112%, 80-125%, and 78-124%, respectively. Furthermore, the quantitation of enrofloxacin obtained from the ELISA and from high-performance liquid chromatography (HPLC) was in good agreement, with the linear regression coefficient between 0.933 and 1.056. The cDNAs encoding a heavy-chain Fd fragment (VH and CH1) and a light chain of the mAb Enro44 were cloned and sequenced. Taken together, the results obtained reveal a potential use of this mAb in an ELISA for the detection of enrofloxacin in food samples. The information of amino acid sequence of this mAb will be useful for further modification and production of the mAb in a bioreactor.

Synthesis of Curcumin Glycosides with Enhanced Anticancer Properties Using One-Pot Multienzyme Glycosylation Technique

  • Gurung, Rit Bahadur;Gong, So Youn;Dhakal, Dipesh;Le, Tuoi Thi;Jung, Na Rae;Jung, Hye Jin;Oh, Tae Jin;Sohng, Jae Kyung
    • Journal of Microbiology and Biotechnology
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    • v.27 no.9
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    • pp.1639-1648
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    • 2017
  • Curcumin is a natural polyphenolic compound, widely acclaimed for its antioxidant, anti-inflammatory, antibacterial, and anticancerous properties. However, its use has been limited due to its low-aqueous solubility and poor bioavailability, rapid clearance, and low cellular uptake. In order to assess the effect of glycosylation on the pharmacological properties of curcumin, one-pot multienzyme (OPME) chemoenzymatic glycosylation reactions with UDP-${\alpha}-{\text\tiny{D}}$-glucose or UDP-${\alpha}-{\text\tiny{D}}$-2-deoxyglucose as donor substrate were employed. The result indicated significant conversion of curcumin to its glycosylated derivatives: curcumin 4'-O-${\beta}$-glucoside, curcumin 4',4"-di-O-${\beta}$-glucoside, curcumin 4'-O-${\beta}$-2-deoxyglucoside, and curcumin 4',4"-di-O-${\beta}$-2-deoxyglucoside. The products were characterized by ultra-fast performance liquid chromatography, high-resolution quadruple-time-of-flight electrospray ionization-mass spectrometry, and NMR analyses. All the products showed improved water solubility and comparable antibacterial activities. Additionally, the curcumin 4'-O-${\beta}$-glucoside and curcumin 4'-O-${\beta}$-2-deoxyglucoside showed enhanced anticancer activities compared with the parent aglycone and diglycoside derivatives. This result indicates that glycosylation can be an effective approach for enhancing the pharmaceutical properties of different natural products, such as curcumin.

Retinol, α-tocopherol, and selected minerals in breast milk of lactating women with full-term infants in South Korea

  • Kim, Hyesook;Jung, Byung-Mun;Lee, Bum-Noh;Kim, Yun-Je;Jung, Ji A;Chang, Namsoo
    • Nutrition Research and Practice
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    • v.11 no.1
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    • pp.64-69
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    • 2017
  • BACKGROUND/OBJECTIVES: This study was performed to measure fat-soluble vitamins and minerals in breast milk of Korean lactating mothers who exclusively breastfed their babies. SUBJECTS/METHODS: Breast milk samples were collected from 334 mothers. Concentrations of retinol and ${\alpha}$-tocopherol were analyzed by high performance liquid chromatography ultraviolet spectrometry while concentrations of minerals were measured by inductively coupled plasma optical emission spectrometry. RESULTS: Retinol and ${\alpha}$-tocopherol contents of breast milk were $39.58{\pm}19.64{\mu}g/dL$ and $0.23{\pm}0.13mg/dL$, respectively. Average sodium, potassium, calcium, phosphorus, and magnesium levels in breast milk were $11.11{\pm}5.16$, $38.56{\pm}9.01$, $27.87{\pm}6.10$, $13.56{\pm}3.30$, and $3.05{\pm}0.65mg/dL$, respectively. Contents of trace elements such as iron, zinc, copper, and manganese were $40.26{\pm}46.21$, $98.40{\pm}62.47$, $24.09{\pm}9.03$, and $0.90{\pm}1.63{\mu}g/dL$, respectively. Fat-soluble vitamin concentration was positively correlated with total fat in milk samples, but no significant differences were observed in levels of retinol, ${\alpha}$-tocopherol, or minerals based on whether or not lactating women were taking dietary supplements. CONCLUSIONS: Micronutrient contents of breast milk samples from Korean lactating women were comparable to those of other nations. Retinol and ${\alpha}$-tocopherol levels were correlated and also with total fat in breast milk.

Residue Analysis of Quinclorac in Soil by Supercritical Fluid Extraction and Fluorogenic Derivatization Coupled with High Performance Liquid Chromatography (초임계추출과 형광유도체를 이용한 HPLC 에서의 Quinclorac 의 토양중 잔류분석)

  • Kim, Yong-Whan
    • Applied Biological Chemistry
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    • v.40 no.5
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    • pp.442-446
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    • 1997
  • A new analytical method was developed by HPLC after supercritical fluid extraction and fluorogenic derivatization for the determination of quinclorac (3,7-dichloro-8-quinoline carboxylic acid) in soil. The graminicide quinclorac was extracted from soil by supercritical fluid extraction. Supercritical carbon dioxide at 7000 psi $(80^{\circ}C)$ modified with 30% of methanol extracted quinclorac from soil samples at the level of $0.1ng\;g^{-1}$ with 96% recovery. Extracted quinclorac was determined by HPLC as a fluorescent derivative. Derivatization was made with 4-bromomethyl-7-methoxycoumarin (4-Br-Mmc) using 18-crown-6-ether as a catalyst. The conversion was completed within 30 min and the limit of detection was 0.5 ppb to prove that the procedure could be used in the residue analysis of the pesticides containing carboxylic acid group.

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Saponin pattern of Panax ginseng root in relation to stem color (경색도별(莖色度別) 고려임삼근(高麗人蔘根)의 사포닌 양상(樣相))

  • Park, Hoon;Parklee, Qwi-Hee;Lee, Chong-Hwa
    • Applied Biological Chemistry
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    • v.23 no.4
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    • pp.222-227
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    • 1980
  • Ginsenosides in two parts (central fart and epidermis-cortex) of main body of Korea ginseng root (purple stem variety) were analyzed by high performance liquid chromatography in relation to purple color intensity on stem. Pattern similarity of saponin by simple correlation of ginsenosides between the same or different parts of root in the same or different group showed that stem color was not associated with saponin pattern in two parts. Saponin pattern was slightly different between different parts regardless of stem color. The order of each ginsenoside content was $Rg_1>Re>Rb_1>Rb_2>Rc>Rg_2{\geq}Rd>Rf$ in epidermis-cortex while $Rg_1>Re{\geq}Rg_2{\geq}Rb_1{\gg}Rb_2>Rc{\geq}Rd>Rf$ in central part.

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Purification of Vasopressin-related Peptide, [Arg8]-vasotocin, from the Brain of Conger Eel (Conger myriaster) (붕장어 (Conger myriaster)의 뇌로부터 Vasopressin-related Peptide, [Arg8]-vasotocin의 정제)

  • Go Hye-Jin;KIM Chan-Hee;KIM Eun Jung;KIM In Hye;PARK Hee Yeon;YOON Ho Dong;HONG Yong-Ki;PARK Nam Gyu
    • Korean Journal of Fisheries and Aquatic Sciences
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    • v.38 no.1
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    • pp.6-11
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    • 2005
  • Vasopressin (VP)-related peptide was purified from the brain extract of conger eel (Conger myriaster) by reverse-phase, ion-exchange high performance liquid chromatography (HPLC). This peptide with a molecular weight of 1,051.2 Da was determined as $H-Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Arg-Gly-NH_2$, whose Cys residues made an intramolecular disulfide bridge by the automated amino acid sequence analysis, MALDI- TOF mass spectrometry. It's sequence was confirmed by identity of the elution position with the synthetic peptide in HPLC system. As a result of homology investigation, the primary structure of this peptide was the same as that of VP-superfamily member, $[Arg^8]-vasotocin$. The synthetic peptide showed a contractile activity at a minimal effective concentration of $10^{-10}\;M$ on the intestinal smooth muscle of goldfish.

Simultaneous Determination of Three Compounds in Ejung Tang by HPLC-DAD and LC-ESI-MS (HPLC를 이용한 이중탕 중 3종 활성성분의 동시분석법 확립)

  • Lee, Bo-Hyoung;Ma, Jin-Yeul;Weon, Jin-Bae;Yang, Hye-Jin;Yun, Bo-Ra;Ma, Choong-Je
    • Korean Journal of Pharmacognosy
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    • v.43 no.1
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    • pp.10-15
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    • 2012
  • An accurate and sensitive analysis method was established for simultaneous determination of three bioactive compounds (glycyrrhizin, 6-gingerol and ginsenoside Rg3) in the Ejung Tang with high-performance liquid chromatography (HPLC)-photodiode array detection (DAD)-electrospray ionization (ESI)-Mass spectrometry (MS). The optimizing chromatographic separations a were acquired by an $C_{18}$ column ($5{\mu}m$, $4.6I.D{\times}250mm$, SHISHEDO) using gradient elution with water comprising 0.1% TFA(trifluoroacetic acid) and acetonitrile at a performing temperature of $35^{\circ}C$. Flow rate was 1.0 ml/min. A detection UV wavelength set at 205 nm and 250 nm. The three compounds were identified by electrospray ionization mass spectrometry. All calibration curves indicated great linear regression within test ranges ($R^2>0.9997$). The established method provided acceptable precision and accuracy. The relative standard deviations (RSDs) of intra-day and inter-day were less than 2.00% and 3.00%, respectively. The recoveries were found to range from 94.49 to 101.10% for the three compounds analyzed. These results showed that this method was effective and reliable for quality control of Eiung-Tang.

Quantitative Analysis of Anthraquinones in Cassiae Semen by Processing Method (수치에 따른 결명자 주요 Anthraquinone의 함량분석)

  • Seo, Chang-Seob;Kim, Jung-Hoon;Shin, Hyeun-Kyoo;Hwang, Seock-Yeon;Kim, Byoung-Soo
    • Korean Journal of Pharmacognosy
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    • v.45 no.3
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    • pp.200-208
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    • 2014
  • In this study, we performed quantification determination of four major components including aurantio-obtusin, emodin, chrysophanol, and physcion in the 70% ethanol extracts of non-processed Cassiae Semen and processed Cassiae Semen using a high-performance liquid chromatography coupled with photodiode array detector. The analytical column for separation of the 4 constituents used a Gemini $C_{18}$ column kept at $40^{\circ}C$ by the gradient elution with 1.0% (v/v) acetic acid in water and 1.0% (v/v) acetic acid in acetonitrile as mobile phase. The flow rate was 1.0 mL/min and the injection volume was $10{\mu}L$. The amount of aurantio-obtusin, emodin, chrysophanol, and physcion in non-processed Cassiae Semen were 0.07%, 0.02%, 0.25%, and 0.10%, respectively. The amount of aurantio-obtusin, emodin, chrysophanol, and physcion in processed Cassiae Semen were 0.04-0.14%, 0.01-0.03%, 0.02-0.42%, and 0.01-0.24%, respectively. Consequently, the optimal processing condition of Cassiae Semen for the improvements of amounts of four anthraquinone compounds was obtained by roasting at $240^{\circ}C$ for 15 min.

Simultaneous Analysis of Bioactive Metabolites from Rehmannia glutinosa by HPLC-DAD-MS/MS

  • Won, Tae-Hyung;Ryu, Sung-Kwang;Kang, Sam-Sik;Shin, Jong-Heon
    • Natural Product Sciences
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    • v.16 no.2
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    • pp.116-122
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    • 2010
  • A high-performance liquid chromatography (HPLC) with diode array detector (DAD) and electrospray ionization mass spectrometry (ESI-MS) was established for the simultaneous determination of five representative metabolites of the iridoid and phenolic classes from Rehmannia glutinosa. The optimal chromatographic conditions were obtained on an ODS column (5 mm, $4.6{\times}250\;mm$) with the column temperature at $25^{\circ}C$. The mobile phase was composed of water and acetonitrile using a gradient elution with the flow rate 0.3 mL/min. Detection wavelength was set at 205 nm. All calibration curves showed good linear regression ($r^2$ > 0.997) within test ranges. Limits of detection (LOD) and quantitation (LOQ) values were lower than 0.123 and $0.373\;{\mu}g/mL$, respectively. The developed method provided satisfactory precision and accuracy with overall intra-day and inter-day variations of 0.09 - 0.76% and 0.16 - 1.41%, respectively, and the overall recoveries of 99.03 - 102.67% for all of the compounds analyzed. In addition, effectiveness of diverse extraction methods was compared to each other for the development of standard analytic method. The verified method was successfully applied to the quantitative determination of five representative metabolites in twenty-one commercial Rehmannia glutinosa samples from different markets in Korea and China. The analytical results showed that the contents of the five analytes vary significantly with sources.

Quantitative Analysis of Icariside E5 and Vanilloyl Icariside E5 from the Seed of Capsicum annuum. L (고추씨로부터 Icariside E5와 Vanilloyl Icariside E5의 함량분석)

  • Youn, Sung Hye;Yin, Jun;Ahn, Hye Shin;Tam, Le Thi;Kwon, Suk Hyung;Min, Bokkee;Yun, Seong Ho;Kim, Hwa Yeon;Lee, Min Won
    • Korean Journal of Pharmacognosy
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    • v.48 no.2
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    • pp.160-165
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    • 2017
  • Capsicum annuum L. (CA) is reported to be the most widely cultivated species of the Capsicum throughout the world. Its fruit and the seeds have been used as spice after drying and grinding. It is reported that the fruit and seeds contain capsaicinoid such as capsaicin and dihydrocapsaicin, carotenoid such as capsanthin and ${\beta}$-carotene with its anti-tumor effect, anti-obesity effect, anti-diabetic action. Present study, we isolated two lignans, icariside $E_5$ and vanilloyl icariside $E_5$ from the seed of CA and validation and content determination were confirmed using High-Performance Liquid Chromatography (HPLC). As a result, the contents of the lignans, icariside $E_5$ and vanilloyl icariside $E_5$ were 0.28%, 0.085%, respectively in the extract.