• 제목/요약/키워드: Alternative assay method

검색결과 114건 처리시간 0.024초

지방산대사이상질환 스크리닝을 위한 TBDMS 유도체화 후 GC-MS를 이용한 혈장 중 유리지방산의 분석 (A Sensitive Determination of Plasma Free Fatty Acids Following Tert-butyldimethylsilyl Derivatization using Gas Chromatography-Mass Spectrometry for Screening of Fatty Acid Oxidation Disorders)

  • 윤혜란;타파마헤샬
    • 대한유전성대사질환학회지
    • /
    • 제17권2호
    • /
    • pp.39-47
    • /
    • 2017
  • Purpose: A sensitive gas chromatography mass spectrometry (GC-MS) method was developed for screening of fatty acid oxidation disorders. Methods: The assay utilized a simple protein precipitation with sulfosalicylic acid followed by tert-butyl dimethylsilyl (TBDMS) derivatization of hydroxyl functional group by N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide (MTBSTFA). Results: Calibration curves of spiked pooled plasma showed a linear relationship in the range of 0.01 ng -2 mg with correlation coefficient value greater than 0.98. Limits of detection (LOD) and limits of quantification (LOQ) were found in the range of 0.9-8.8 ng and 9-88 ng, respectively. Conclusion: The new developed method might be useful for a rapid, sensitive screening of inherited fatty acid oxidation disorders. In addition, the method expected to be one of the alternative method for screening newborns of metabolic disorders in the laboratories where expensive MS/MS is unavailable.

  • PDF

Picomolar Scale Determination of Carbohydrates Covalently Immobilized on Activated Beads Using Hydroxyl Functionality

  • Yu, Jae-Hoon;Chun, Sung-Min;Park, Ho-Koon;Park, Yong-Keun;Jeong, Sun-Joo
    • BMB Reports
    • /
    • 제32권1호
    • /
    • pp.98-102
    • /
    • 1999
  • Since carbohydrates are major mediators in cell-to-cell adhesion and communication, the development of specific and strong binders against them could generate promising therapeutics. As the first step towards that goal, sugar molecules have to be immobilized to be used as an affinity matrix. The amino functionality in sugar is the most active nucleophile for the immobilization, if the amino group is available. An alternative and general method is to use the hydroxyl group as a direct nucleophile, but the quantitation of immobilized hydroxyl groups is not easily done. To overcome this limitation, we have developed a method to immobilize various isomers of monosaccharides with p-nitrophenyl groups to the beads by using their hydroxyl groups. It was found that the amount of immobilized sugar was independent of the structure of the sugar, but was dependent on the number of hydroxyl groups. We also developed a sensitive method to quantify the amount of immobilized sugar at the picomolar scale by utilizing commercially available glycosidases to release a sensitive reporter molecule, p-nitrophenol, and detect it by HPLC. This new technique would allow a facile quantitation method for immobilized sugar molecules, which could be used as the affinity matrix to develop strong binders against biologically important sugars.

  • PDF

감마선 조사된 저분자 laminarin의 항산화 활성 연구 (Antioxidant Activity of Low Molecular Weight Laminarin Prepared with Gamma Irradiation)

  • 최종일;김현주;이주운
    • KSBB Journal
    • /
    • 제26권6호
    • /
    • pp.565-568
    • /
    • 2011
  • In this study, it was investigated the antioxidant activity of laminarin degraded by gamma irradiation. Because the activities of antioxidants have been attributed to various mechanisms, different assay methods have been conducted and compared. 1,1-diphenyl-2-picryl-hydrazyl (DPPH) radical scavenging activity, ferric reducing antioxidant power, and lipid peroxidation inhibitory activity of degraded laminarin were measured and compared with non-degraded. All of these results showed that the antioxidant activity of laminarin degraded by irradiation was increased depending on the absorbed dose. Therefore, gamma irradiation could be an alternative method for the preparation of degraded laminarin with higher antioxidant activity.

Phototoxicity: Its Mechanism and Animal Alternative Test Methods

  • Kim, Kyuri;Park, Hyeonji;Lim, Kyung-Min
    • Toxicological Research
    • /
    • 제31권2호
    • /
    • pp.97-104
    • /
    • 2015
  • The skin exposure to solar irradiation and photoreactive xenobiotics may produce abnormal skin reaction, phototoxicity. Phototoxicity is an acute light-induced response, which occurs when photoreacive chemicals are activated by solar lights and transformed into products cytotoxic against the skin cells. Multifarious symptoms of phototoxicity are identified, skin irritation, erythema, pruritis, and edema that are similar to those of the exaggerated sunburn. Diverse organic chemicals, especially drugs, are known to induce phototoxicity, which is probably from the common possession of UV-absorbing benzene or heterocyclic rings in their molecular structures. Both UVB (290~320 nm) and UVA (320~400 nm) are responsible for the manifestation of phototoxicity. Absorption of photons and absorbed energy (hv) by photoactive chemicals results in molecular changes or generates reactive oxygen species and depending on the way how endogenous molecules are affected by phototoxicants, mechanisms of phototoxcity is categorized into two modes of action: Direct when unstable species from excited state directly react with the endogenous molecules, and indirect when endogeneous molecules react with secondary photoproducts. In order to identify phototoxic potential of a chemical, various test methods have been introduced. Focus is given to animal alternative test methods, i.e., in vitro, and in chemico assays as well as in vivo. 3T3 neutral red uptake assay, erythrocyte photohemolysis test, and phototoxicity test using human 3-dimensional (3D) epidermis model are examples of in vitro assays. In chemico methods evaluate the generation of reactive oxygen species or DNA strand break activity employing plasmid for chemicals, or drugs with phototoxic potential.

Alternative chromatographic method for the assay test of terbutaline and salbutamol using ionic liquid assisted aqueous mobile phase

  • Mai, Xuan-Lan;Choi, Yusung;Truong, Quoc-Ky;Nguyen, Thi-Ngoc-Van;Han, Sang Beom;Kim, Kyeong Ho
    • 분석과학
    • /
    • 제33권4호
    • /
    • pp.169-176
    • /
    • 2020
  • Separation of basic compounds using reverse phase chromatography on a silica-based stationary phase represents a major challenge, because of the interaction between the cationic sites of the basic compounds with the anionic silanols of the stationary phase. This study presents a simple, reliable, and organic solvent - free liquid chromatographic method for the determination of terbutaline and salbutamol, in which a room temperature ionic liquid (RTIL) is used as mobile phase additive. We investigated various mobile phase parameters affecting the retention of the two compounds, such as types and concentration of RTILs and, pH of the mobile phase were investigated. The developed method was validated according to International Conference on Harmonization (ICH) guidelines and successfully applied effectively to determine salbutamol sulfate in pharmaceutical preparations.

Identification of Lactic Acid Bacteria in Kimchi Using SDS-PAGE Profiles of Whole Cell Proteins

  • Kim, Tae-Woon;Jung, Sang-Hoon;Lee, Ji-Yeon;Choi, Sun-Kyu;SUN-HEE-PARK;JAE-SUN-JO
    • Journal of Microbiology and Biotechnology
    • /
    • 제13권1호
    • /
    • pp.119-124
    • /
    • 2003
  • This study was conducted to evaluate the practical usefulness of the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PACE) fingerprinting of whole cell proteins far the identification of lactic acid bacteria in Kimchi. SDS- PACE of whole cell proteins of the reference strains and lactic acid bacteria isolated from Kimchi yielded differential banding patterns that were highly specific fingerprints, thus making it possible to identify. Identification of the isolates from Kimchi was achieved by comparing the SDS-PAGE fingerprints of isolates to those of reference strains. In addition, the reliability of SDS-PAGE was examined by comparing the results with those of the APL 50 CHL system assay and 16S rRNA gene sequence. SDS-PACE assay showed a different identity to reference strains, while the APL 50 CHL system and 16S rRNA gene sequence could not distinguish a few strains. Therefore, SDS-PAGE of the whole cell proteins is a specific and a reliable method that will be useful for the identification of lactic acid bacteria in Kimchi to the species level, and can be used as an alternative or complementary identification method.

고성능액체크로마토그래피에 의한 Isothiazolinone Components의 분석에 관한 연구 (A Study on the Analysis of Isothiazolinone Components by High Performance Liquid Chromatography)

  • 김종규;이덕희
    • 한국환경보건학회지
    • /
    • 제18권2호
    • /
    • pp.102-105
    • /
    • 1992
  • 냉각탑, 제지산업, 일반 산업용수 등에 사용되는 산업용 방부제의 유효 성분 isothiazolinone components 즉, 2-methyl-4-isothiazolin-3-one(unchlorinated compound)과 5-chloro-2-methy14-isothiazolin-3-one(chlorinated compound)을 고성능액체크로마토그래피로서 분리, 정량하였다. 역상의 C$_{18}$ column (15 cmX3.9 mm I.D.)을 사용하였으며 자외선 검출기의 파장 254 nm에서 methanol-0.4% acetic acid(40 : 60)의 이동상, methanol-0.4% acetic acid(30: 70)의 추출 및 주입용매 조건으로 HPLC 분리를 시도한 결과 unchlorinated compound는 10~32,400 mg/l 범위에서, 그리고 chlorinated compound는 120~107,400 mg.l의 범위에서 직선성을 보였다. 내부 표준물질로서 dimethyl phthalate를 사용하였으며, unchlorinated compound, chlorinated compound 및 내부 표준물질의 순서로 븐리가 일어났고 총 분리시간은 6.41분이었다. 상기의 조건으로 시험물질을 분석, 정량한 결과 예측농도치에 근접한 수치를 얻었다.

  • PDF

Heterosigma akashiwo를 모니터하기 위한 뉴클레아제 보호 분석이 통합된 샌드위치 혼성(NPA-SH)의 개발 (Development of a Nuclease Protection Assay With Sandwich Hybridization (NPA-SH) to Monitor Heterosigma akashiwo)

  • 강민경;박미례;김강은;이택견
    • 생명과학회지
    • /
    • 제30권1호
    • /
    • pp.26-31
    • /
    • 2020
  • Heterosigma akashiwo는 전세계적으로 분포된 침편모조류이며, 대발생을 형성하여 많은 나라의 양식산업에 커다란 손실을 유발시킨다. 따라서 빠르고 민감한 검출방법을 개발하는 것은 유해조류 대발생에 대한 적절한 경보를 위해서 필요하다. 이 연구에서는 H. akashiwo를 정성 및 정량적으로 검출하기 위하여 뉴클레아제 보호 분석이 통합된 샌드위치 혼성(NPA-SH)을 개발하였다. NPA-SH 프로브는 여섯 종의 미세조류 핵산 서열을 이용하여 디자인 후, 특이성을 확인하여 capture 프로브와 signal 프로브를 선정하였다. 배양시료와 현장시료를 이용하여 NPA-SH의 적용성을 평가한 결과, NPA-SH의 좋은 적용성과 효과를 확인하였다. H.akashiwo의 정량분석을 위한 선형회귀식을 확인하였으며, 최소 검출한계는 1×104 cells/ml이었다. NPA-SH를 사용하여 얻은 H. akashiwo의 정량결과와 현미경을 사용하여 얻은 결과 사이에는 통계학적으로 유의한 차이는 없었다. NPA-SH 분석은 환경시료에 잘 적용되었다. 이러한 결과는 NPA-SH가 H. akashiwo의 모니터링에 사용되어 왔던 전통적인 현미경적 방법에 대한 좋은 대안이 될 수 있음을 보여준다.

Determination of Free Amino Acids in Isatidis Radix By HILIC-UPLC-MS/MS

  • Pan, Yilin;Li, Jin;Li, Xiang;Chen, Jianwei;Bai, Ganggang
    • Bulletin of the Korean Chemical Society
    • /
    • 제35권1호
    • /
    • pp.197-203
    • /
    • 2014
  • A rapid, accurate and precise method for the determination of 22 amino acids in Isatidis Radix by Hydrophilic Interaction Ultra-High-Performance Liquid Chromatography Coupled with Triple-Quadrupole Mass Spectrometry (HILIC-UPLC-MS/MS) was established. Chromatographic separation was carried out on a Acquity UPLC BEH Amide column ($2.1mm{\times}100mm$, $1.7{\mu}m$) with gradient elution of acetonitrile (containing 0.05% formic acid and 2 mM ammonium formate) and water (containing 0.15% formic acid and 10 mM ammonium formate) at a flow rate of 0.4 mL/min; Waters Xevo$^{TM}$ TQ worked in multiple reaction monitoring mode. All components were separated in 17 min. All calibration curves were linear ($R^2$ > 0.991) over the tested ranges. The limits of detection (LOD) and limits of quantitation (LOQ) for these compounds were 0.21-79.55 and 0.72-294.23 ng/mL, respectively. The average recoveries were in the range of 93.75-104.16% with RSD value less than 6.56%. Therefore, this method could be an alternative assay for the determination of 22 amino acids in Isatidis Radix due to its rapidness, sensitivity, less sample and solvent consumption.