• Title/Summary/Keyword: Normal phase HPLC

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Mobile Phase Compositions for Ceramide III by Normal Phase High Performance Liquid Chromatography

  • Hong, Seung-Pyo;Lee, Chong-Ho;Kim, Se-Kyung;Yun, Hyun-Shik;Lee, Jung-Heon;Row, Kyung-Ho
    • Biotechnology and Bioprocess Engineering:BBE
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    • v.9 no.1
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    • pp.47-51
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    • 2004
  • Ceramide III was prepared by the cultivation of Saccharomyces cerevisiae. Ceramide III was partitioned from the cell extracts by solvent extraction and analyzed by Normal Phase High Performance Liquid Chromatography (NP-HPLC) using Evaporative Light Scattering Detector (ELSD). We experimentally determined the mobile phase composition to separate ceramide III with NP-HPLC. Three binary mobile phases of n-hexane/ethanol, n-hexane/lsoprophyl Alcohol(IPA) and n-hexane/n-butanol and one ternary mobile phase of n-hexane/IPA/methanol were demonstrated. For the binary mobile phase of n-hexane/ethanol, the first mobile phase composition, 95/5(v/v), was step-increased to 72/23(v/v) at 3 min. In the binary mobile phase, the retention time of ceramide III was 7.87min, while it was 4.11 min respectively in the ternary system, where the mobile phase composition of n-hexane/IPA/methanol, 85/7/8(v/v/v), was step-increased to 75/10/15(v/v/v) at 3 min. However, in the ternary mobile phase, the more peak area of ceramide III was observed.

Purification and Characterization of Paclitaxel from Plant Cell Cultures of Taxus chinensis in Large-Scale Process (식물세포 Taxus chinensis 배양으로부터의 Paclitaxel 대량 정제 및 특성)

  • 김진현;기은숙;민범찬;최형균;홍승서;이현수
    • KSBB Journal
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    • v.15 no.5
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    • pp.537-540
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    • 2000
  • In developing a HPLC purification process, it was hoped that a single chromatographic system would be sufficient to abtain pure paclitaxel in high yield. However, no such system was found, due in part to the complex taxoid profile of crude paclitaxel and to the rigorous nature of the product specification. A two step HPLC purification was adopted using reverse-phase separation on C(sub)18 as a first step, and normal-phase separation on silica as the final polishing step. Impurity profiles were established and maintained for paclitaxel, which identified and quantified each impurity observed in purified paclitaxel from these two steps, all impurities at or above 0.1% were identified. Results provide information for improving the quality control of paclitaxel production.

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Determination of Putrescine, Spermidine and Spermine in urine by HPLC (HPLC법에 의한 뇨 중의 Putrescine, Spermidine 및 Spermine의 정량)

  • Shin, Tae-Yong;Lee, Seok-Bong;Kim, Hyung-Min
    • Analytical Science and Technology
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    • v.10 no.6
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    • pp.445-452
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    • 1997
  • A simple and sensitive high performance liquid chromatography for analysis of Putrescine, Spermidine and Spermine in urine is described. The benzoyl chloride derivatives of Putrescine, Spermidine and Spermine are separated on a ${\mu}$ Bondapak $C_{18}$ reverse-phase column with methanol/$H_2O$(50/50v/v) as the mobile phase. The results show that the mean level of polyamines in uterine cancer patients urine is much higher than in normal human urine.

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Determination of Polyamines in Serum Using Benzoyl Chloride as Derivatization Agent by HPLC (고속액체크로마토그래피에 의한 벤조일 클로라이드를 유도체화제로 한 혈청 중의 폴리아민의 분리 정량)

  • Lee, Seok-Bong;Ko, Yong-Seok;Kim, Hyung-Min;Moon, Young-Hoe;Jin, Weon-Taek;Shin, Tae-Yong
    • YAKHAK HOEJI
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    • v.41 no.4
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    • pp.414-420
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    • 1997
  • A rapid and sensitive high performance liquid chromatographic method with UV detection for the analysis of polyamines(putrescine, spermidine, spermine) has been developed. The benzoyl chloride derivatives of putrescine, spermidine and spermine are separated on a mc-Bondapak C18 reverse-phase column with 50% methanol as the mobile phase. This method was applied to the analysis of polyamines in the serum of normal human and uterine cancer patients. The results show that the mean level of polyamines in cancer patients serum is much higher than that in normal human serum.

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Reverse-Phase HPLC Method for Identification of Diastereomeric Constituents from Sasa borealis (Sasa borealis의 Diastereomeric 성분들의 역상 고속액체크로마토그래프 분석방법)

  • Jeong Yeon Hee;Lee Jun;Kwon Youngjoo;Seo Eun-Hyoung
    • YAKHAK HOEJI
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    • v.50 no.1
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    • pp.21-25
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    • 2006
  • Reiterated normal-phase column chromatography lead to the isolation and purification of six known compounds but for the first time from the whole plant of Sasa borealis (Hack.) Makino (Gramineae): tricin 4'-O-(erythro-${\beta}$-guaia-cylglyceryl) ether (1), tricin 4'-O-(threo-${\beta}$-guaiacylglyceryl) ether (2), tricin 4'-O-[erythro-${\beta}$-guaiacyl-(9'-O-acetyl)-glyceryl] ether (3), tricin 4'-O-[threo-${\beta}$-guaiacyl-(9'-O-acetyl)-glyceryl] ether (4), (-)-pinoresinol (5), and vanillin (6). The structures of the compounds (1-6) were established based on interpretation of high resolution NMR (COSY, HSQC, HMBC, and NOESY) spectral data. In particular, compounds 1 and 3 were diastereomers of compounds 2 and 4, respectively. These two sets of diastereomers were able to be simultaneously identified and quantified by a gradient reversed-phase HPLC method with UV photodiode array, This sensitive HPLC method is noteworthy as a simultaneous separation and identification method to test the extract of the family Gramineae which contains these compounds.

Absence of Tocotrienol Form of Vitamin E in Purple Perilla (Perilla frutescens var. acuta Kudo) Seeds Confirmed by Comparative Analysis Using HPLC and GC (GC 및 HPLC 비교분석에 기초한 차조기 종실내 tocotrienol 부재의 평가)

  • Lee, Young-Sang;Kim, Min-Kyoung
    • KOREAN JOURNAL OF CROP SCIENCE
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    • v.53 no.spc
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    • pp.115-120
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    • 2008
  • Lipid soluble vitamin E consists of tocopherols and tocotrienols depending upon double bonds in phytyl side chains attached to chromanol ring. Recent reports on antioxidative, anticancer, and cholesterol-lowering effects of tocotrienols have increased researches and commercialization of tocotrienols. Purple perilla (Perilla frutescens var. acuta Kudo) has been reported as a plant containing tocotrienols along with tocopherol forms of vitamin E based upon normal phase HPLC analysis. To confirm the existence or absence of tocotrienol form of vitamin E in purple perilla, comparative analysis using HPLC, GC/FID, and GC/MSD has been conducted for 14 purple perilla genetic accessions collected from Korea and Japan. Normal phase HPLC analysis showed ${\alpha}-$, ${\beta}-$, ${\gamma}-$, and ${\delta}-tocopherols$ along with peaks with retention times quite similar to ${\beta}-$ and ${\gamma}-tocotrienols$. Same purple perilla samples, analysed by GC exhibited ${\alpha}-$, ${\beta}-$, ${\gamma}-$, and ${\delta}-tocopherols$ quantitatively equivalent to HPLC results. However, no peaks for ${\beta}-$ and ${\gamma}-tocotrienols$ could be observed and unknown two peaks of similar retention times with ${\beta}-$ and ${\gamma}-tocotrienols$ were identified not corresponding tocotrienols by GC/MSD. These results suggest the absence of tocotrienol form of vitamin E in purple perilla as well as the necessity of using GC-based qualitative and quantitative vitamin E analysis to avoid misinterpretation of peaks with similar retention times as tocotrienol isomers when analysed by an HPLC.

Investigation of brassinosteroid substances in the shoots of japonica type Korean rice (일반계 벼의 brassinosteroid활성물질 검색)

  • Hyun, Kyu-Hawn;Park, Keun-Hyung
    • Applied Biological Chemistry
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    • v.33 no.4
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    • pp.307-314
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    • 1990
  • Brassinosteroid-like substances in two japonica type Korean rices were investigated. The extracts from the shoots at the maximum tillering stage were purified by solvent fractionation, silica gel adsorption chromatography, Sephadex LH-20 chromatography, charcoal adsorption chromatography, preparative TLC, Bondesil chromatography and HPLC of normal phase and reverse phase, successively. Biological activity of each purification step were monitored by the rice lamina inclination test. Two cultivars tested in this experiment produced brassinosteroids and endogenous brassinosteroids showing similiarity between two cultivars.

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HPLC Analysis of Piroxicam in the Rabbit Plasma and its Bioavailability after the Transdermal Administration of Patches (토끼 혈장 중 피록시캄의 HPLC 분석 및 패취제 투여 후 경피흡수)

  • Shin, Dae-Hwan;Park, Seong-Hyeok;Lee, Gyeong-Bok;Lee, Chong-Kil;Chung, Youn-Bok
    • Journal of Pharmaceutical Investigation
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    • v.39 no.3
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    • pp.177-183
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    • 2009
  • A rapid and sensitive reversed-phase high performance liquid chromatography (HPLC) method was developed for the determination of piroxicam in the rabbit plasma. After a treatment of plasma sample by liquid-liquid extraction, the drug was analyzed on an HPLC system with ultraviolet detection at 330 nm. HPLC was carried out using reversed-phase isocratic elution with a C18 column, a mobile phase of a mixture of acetonitril, doubly deionized water and acetic acid 43.74:56.00:0.26 v/v%) at a flow rate of 1.1 mL/min. The chromatograms showed good resolution and sensitivity and no interference of plasma. The calibration curve for the drug in plasma sample was linear over the concentration range of 0.01-2.0 ${\mu}$g/mL. The intra- and inter-day assay accuracies of this method ranged from 86.82% to 108.33% of normal values and the precision did not exceed 13% of relative standard deviation. The plasma concentration of piroxicam decreased to below the quantifiable limit at 12 hr after the i.v. bolus administration to rabbits following dose of 0.375 mg/kg yielding a apparen t plasma half life of 1.38 hr. The transdermal route prolongs plasma levels of piroxicam. The bioavailability, calculated from the dose-adjusted ratio of the $AUC_{transdermal}$ to the $AUC_{i.v.}$, was 7.44%. The plasma concentration of piroxicam was detected by 48 hr after the transdermal administration of patch at a dose of 32 mg/kg. This method was suitable for cutaneous absorption studies of piroxicam in the rabbit after transdermal administration of different types of dosages of the drug.

Quantitation of Mevinolinic Acid in Human Plasma by HPLC (고속액체크로마토그래피를 이용한 사람 혈장중 메비놀린 산의 정량)

  • Oh, Han-Suk;Park, Dong-Young;Seo, Sung-Hoon;Kim, Young-Gwan;Hong, Seon-Pyo;Choi, Young-Wook;Lee, Kyung-Tae
    • Journal of Pharmaceutical Investigation
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    • v.30 no.4
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    • pp.279-282
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    • 2000
  • Simple and precise high-performance liquid chromatographic (HPLC) assay was developed and validated for the determination of a HMG-CoA reductase inhibitor, $lovastatin^{TM}$ and its active metabolite (mevinolinic acid) in human plasma. The method involved solid phase extraction of mevinolinic acid and internal standard using Sep-Pak Cartridge. Samples were analyzed by reversed-phase HPLC using $Capcell-Pak\;C_{18}$ column with ultraviolet detection at 238 nm. The quantitation limit of mevinolinic acid was 2 ng/ml and the calibration curve was linear over the range of 2-50 ng/ml $(r^2>0.999)$ with human plasma. The analyses of quality control samples indicated that the normal values could be predicted with an accuracy >97%. The intra- and inter-day coefficients of variation for the analyses were <10%. The average recoveries were similar (79%) for mevinolinic acid and methylmevinolinic acid. The method described has been successfully applied to the quantification of mevinolinic acid in about 1,000 human plasma samples over six-month period.

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