• 대한화학회지
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• 제43권4호
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• pp.438-446
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• 1999

오존처리의 소독 부산물인 알데하이드류를 HPLC로 분석하는 최적 분석법을 확립하였다. 예비실험을 통하여 알데하이드류는 포름알데하이드, 아세트알데하이드, 아크롤레인, 프로피온알데하이드, 부틸알데하이드, 벤즈알데하이드 6종과 케톤류인 아세톤 1종 모두 7종을 대상 시료로 선정하였다. HPLC에 의한 알데하이드-DNPH 유도체를 최적화 하기 위해 시트르산 완충용액의 pH, 반응 온도, 반응 시간, DNPH 농도, 추출 용매의 종류 및 조성의 최적 조건을 구하였다. 그 결과 시트르산 완충 용액의 pH는 3.0, 반응 온도는 40$^{\circ}C$, 반응시간은 15분, DNPH의 농도는 0.012%에서 최적 반응 조건임을 알 수 있었으며, 반응이 완결된 알데하이드-DNPH 유도체를 $C_18$Sep-Pak 카트리지에 농축시켜 과량의 DNPH를 용리시킨 다음 탈착용매로서 THF/ACN=70/30의 혼합용매 2mL로 탈착하였을 때 87∼107% 범위의 회수율을 나타내었다. HPLC 분리 조건으로서 Nova-Pak $C_18$ 컬럼상에서 이동상의 초기 조건은 ACN/MeOH/Water=30/10/60 에서 최종 조건은 80% ACN으로 하는 기울기 용리를 수행하였을 때 7종의 알데하이드류가 20분 이내에 용리되면서 모두 거의 기준선 분리가 되었다. 본 실험에서 확립한 알데하이드-DNPH 유도체의 최적 분석 조건과 EPA Method 554와 회수율을 비교한 결과, 본 실험에서는 86∼103%, EPA Method 554로부터는 84∼103%으로 거의 일치한 결과를 나타내었다.

• Korean Chemical Engineering Research
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• 제49권6호
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• pp.840-845
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• 2011

아가로오스 분해산물은 생리활성이 우수하여 의약품 및 기능성 화장품 원료로 사용될 뿐만 아니라 바이오에탄올 생산을 위한 효모 발효용 기질로 검토되고 있어 상당한 주목을 받고 있다. 이에 따라 고성능 아가레이즈 탐색에 대한 연구가 활발히 진행되고 있다. 최근 본 연구팀에서는 남해안에서 신규 아가레이즈를 생산하는 미생물인 Pseudoalteromonas sp.를 분리하였다. 본 연구에서는 음이온교환수지를 이용하여 미생물 배양액으로부터 아가레이즈를 분리 정제하기 위한 크로마토그래피 조건을 최적화 하였다. 황산암모늄을 첨가하여 배양 상등액으로부터 침전된 단백질을 회수하고 이를 투석하여 조효소액을 얻었다. 음이온 교환수지인 DEAE-Sepharose 레진이 충진된 칼럼에 조효소액을 로딩하여 아가레이즈를 분리하였다. 410 ${\mu}g$의 단백질 로딩 시 흡착에 적합한 평형 pH는 7.5~8.0 이었고, 적정한 resin의 부피는 3 mL였다. 등용매 용출에서 용출액 중의 NaCl 농도가 증가함에 따라 용출되는 단백질 양이 증가하여 400 mM의 NaCl에서 최대에 달하였다. 최종적으로, NaCl 농도를 1,000 mM까지 선형적으로 증가시키며 아가레이즈를 분리하였다. Lugol 용액을 이용한 염색법으로 용리액 중의 아가레이즈의 존재를 확인하였다.

• 분석과학
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• 제20권5호
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• pp.424-433
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• 2007

혈장시료 중의 갑상선 호르몬을 고체상 추출법을 이용하여 추출한 후 HPLC/DAD/ESI-MS (high-performance liquid chromatograph/diode array detector/electrospray ionization-mass spectrometer)를 사용하여 분석하였다. 7종의 thyroid hormones의 HPLC 분리조건은 Hypersil ODS 컬럼(4.6 mm I.D., 250 mm length, particle size $5{\mu}m$)을 사용하고 ammonium formate buffer와 아세토나이트릴을 이동상으로 하여 기울기 용리한 결과 완전 분리가 가능하였으며, UV spectra 및 질량스펙트럼을 확인할 수 있었다. 고체상추출법에 의한 전처리 최적 조건을 조사한 결과 시료를 pH 3으로 한 후 C18 고체상을 사용하여 4 mL의 메탄올로 용리할 경우 회수율이 74.5-115.7%로 나타났다. HPLC/ESI-MS를 이용하여 20-500 ng/mL범위에서 검량선을 작성한 결과 $r^2$값은 0.9939-0.9978으로 나타났으며 검출한계는 20-50 ng/mL (38.1-162.8 pmol/mL)로 계산되었다. 정상인의 혈장에서 thyroxine의 정량이 가능하였으며 그 결과 50.98-112.97 ng/mL로 검출되었다.

• Bulletin of the Korean Chemical Society
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• 제31권5호
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• pp.1159-1164
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• 2010

Several triterpenoid saponins from root of Pulsatilla koreana Nakai (Ranunculaceae) were studied and their biological activities were reported. It is difficult to analyze triterpenoid saponins using HPLC-UV due to the lack of chromophores. So, evaporative light scattering detection (ELSD) is used as a valuable alternative to UV detection. More recently, a charged aerosol detection (CAD) has been developed to improve the sensitivity and reproducibility of ELSD. In this study, we developed and validated a novel method of high performance liquid chromatography coupled with a charged aerosol detector for the simultaneous determination of four triterpenoid saponins: pulsatilloside E, pulsatilla saponin H, anemoside B4 and cussosaponin C. Analytes were separated by the Supelco Ascentis$^{(R)}$ Express C18 column (4.6 mm ${\times}$ 150 mm, 2.7 ${\mu}m$) with gradient elution of methanol and water at a flow rate of 0.8 mL/min at $30^{\circ}C$. We examined various factors that could affect the sensitivity of the detectors, including various concentrations of additives, the pH of the mobile phase, and the CAD range. Linear calibration curves were obtained within the concentration ranges of 2 - 200 ${\mu}g$/mL for pulsatilloside E, anemoside $B_4$ and cussosaponin C, and 5 - 500 ${\mu}g$/mL for pulsatilla saponin H with correlation coefficient ($R^2$) greater than 0.995. The limit of detection (LOD) and quantification (LOQ) were 0.04 - 0.2 and 2 - 5 ${\mu}g$/mL, respectively. The validity of the developed HPLC-CAD method was confirmed by satisfactory values of linearity, intra- and inter-day accuracy and precision. This method could be successfully applied to quality evaluation, quality control and monitoring of Pulsatilla koreana.

• 사상체질의학회지
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• 제18권1호
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• pp.147-154
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• 2006

1. Objectives This study was aimed to evaluate marker substances in Gamijowi-tang by high performance liquid chromatography(HPLC) 2. Methods The seperation was performed an AquasilC18(4.6X250mm)column by gradient elution with 0.1N H3PO4 in H2O - acetonitrile(0min 100:10,20min60:40, 40min 80:20, 60min 100:0) as the mobile phase at a flow-rate of 1.0 ml/min with detection at 190-800nm 3. Results Ephedrine and schizandrin were established as marker substances in Gamijowi-tang and its qualitative analysis was carried out. Analyzed was similarity of marker substances between in standard article and in Gamijowi-tang. The result of the analysis was that ephedrine and schizadrin showed 0.9999 of similarity, which meant they were really ephedrine and schizadrin. In addition, ephedrin content was 12.16mg, pseudoephedrine conrenr was 5.09mg, and schizandrin content was 26.6mg in 76g of Gamijowi-tang. 4. Conclusions Qualitative and quantitative analyses of ephedrine and schizandrin in Gamijowi-tang were conducted, and it was identified thar they could be used as marker substances in standardizing quality of Gamijowi-rang.

• 동의생리병리학회지
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• 제29권2호
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• pp.168-173
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• 2015

Bangpungtongseong-san(BPTSS, Fangfengtongsheng-san in Chineses) is a traditional herbal formula comprising 18 medicinal herbs. In the present study, we performed the simultaneous analysis for four compounds of BPTSS and examined anti-allergic effects in human keratinocytes and mouse splenocytes. The column for separation of four compounds was used Luna C18 column and maintained at 40℃. The mobile phase for gradient elution consisted of two solvent systems. The analysis was carried out at a flow rate of 1.0 mL/min with PDA detection at 254 and 280 nm. To evaluate production and expression of Th2 chemokines, ELISA and RT-PCR were conducted in tumor necrosis factor (TNF)-α and interferon (IFN)-γ-stimulated HaCaT cells with or without BPTSS or silymarin, a positive control for skin inflammation. To measure Th2 cytokines, primary mouse splenocytes were treated with BPTSS and performed ELISA for interleukin (IL)-4, 5, 13. Calibration curves were acquired with r2>0.9999. The contents of geniposide, liquiritin, baicalin, and glycyrrhizin in BPTSS were 5.06 ㎎/g, 7.33 ㎎/g, 27.56 ㎎/g, and 7.81 ㎎/g, respectively. BPTSS reduced TARC and RANTES production and mRNA expression in TNF-α and IFN-γ-treated HaCaT cells. BPTSS inhibited IL-4, 5, and 13 production in mouse splenocytes. Our data will be a helpful information to upgrade quality control and anti-allergic effects of BPTSS.

• 약학회지
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• 제57권5호
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• pp.330-336
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• 2013

"The Act on Medication Treatment of Sexual Impulse of Sex Offenders" known as chemical castration has been effective since July 2011 in Korea. According to the law, monitoring of male sex hormone in urine is enforced to request National Forensic Service more than once a month after injection of medicine designed to reduce sex impulse. We established a rapid and sensitive method for the monitoring of testosterone (T) and epitestosterone (E) in human urine by liquid chromatography with tandem mass spectrometry (LC-MS/MS). Three mL of urine was pretreated by solid-phase extraction for purification and performed enzymatic hydrolysis. The pretreated samples were extracted twice with 2 ml of ethyl acetate and n-hexane (2 : 3). The separation was applied on Thermo Hypersil GOLD C18 column ($1.9{\mu}m$, $100{\times}2.1mm$). A gradient elution of methanol and water of 0.1% formic acid were used as mobile phase and the retention time was less than 10 min. LC-MS/MS system coupled with an electrospray ionization source was performed in multiple reaction monitoring mode. The transitions of the analytes executed as following: m/z $289{\rightarrow}97$, 109 for T and E, m/z $292{\rightarrow}109$ for $T-d_3$ and $E-d_3$ as internal standards. The validation results of the method were satisfactory. The limits of detection were 0.05 ng/ml and the limits of quantification were 0.1 ng/ml. This method was successfully applied to real human urine sample. The developed method will be useful for monitoring T/E ratio in urine of sex offenders.

• 대한예방한의학회지
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• 제17권3호
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• pp.165-176
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• 2013

Objective : The purpose of this study was to establish the simultaneous analysis for six compounds in Gumiganghwal-tang (GMGHT, Jiuweiqianghuo-tang) and to investigate the anti-atherosclerotic effects of GMGHT in vitro. Methods : The column for separation of six compounds was used Luna $C_{18}$ column and maintained at $40^{\circ}C$. The mobile phase for gradient elution consisted of two solvent systems, 1.0% acetic acid in water and 1.0% acetic acid in acetonitrile. The analysis was carried out at a flow rate of 1.0 mL/min with pothodiode array (PDA) detection at 254, 280, and 320 nm. The injection volume was 10 ${\mu}L$. The antioxidant activities of GMGHT were evaluated by measuring free radical scavenging activities on 2,2'-Azinobis-3-ethyl-benzothiazoline-6-sulfonic acid (ABTS) and 1-1-diphenyl-2-picrylhydrazyl (DPPH). The inhibitory effects on low-density lipoprotein (LDL) oxidation were evaluated by the formation of thiobarbituric acid relative substances (TBARS), relative electrophoretic mobility (REM), and fragmentation of apolipoprotein B (ApoB)-100. Results : Calibration curves were acquired with $r^2{\geq}0.9998$. The contents of liquiritin, ferulic acid, baicalin, baicalein, glycyrrhizin, and wogonin in GMGHT were 1.784, 1.693, 37.899, 0.258, 1.869, and 0.034 mg/g, respectively. The GMGHT showed the radical scavenging activity in a dose-dependent manner. The concentration required for 50% reduction ($RC_{50}$) against ABTS and DPPH radicals were 72.51 ${\mu}g/mL$ and 128.49 ${\mu}g/mL$. Furthermore, GMGHT reduced the oxidation properties of LDL induced by $CuSO_4$. Conclusion : HPLC-PDA is considered as an available and convenient method for quality control and standardization of GMGH and GMGHT has potentials on anti-atherosclerosis by anti-oxidative effect and suppressive effect on LDL oxidation.

• Bulletin of the Korean Chemical Society
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• 제26권5호
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• pp.747-754
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• 2005

Liquid chromatographic methods with UV and fluorescence detection have been used to determine the levels of (+)-catechin, (-)-epicatechin, (-)-epicatechin gallate, and (-)-epigallocatechin gallate in Korean green tea infusions. The extracts of Korean green tea leaves or powders in water at various temperatures (100 ${^{\circ}C}$, 80 ${^{\circ}C}$, 60 ${^{\circ}C}$) and time, were washed with chloroform and re-extracted to ethyl acetate. The ethyl acetate phase was dried and re-dissolved in methanol and analyzed. Five catechin compounds were separated by gradient elution. The flavonoids were found decomposed on prolonged extraction, thus exhaustive extraction by a Soxhlet apparatus was found useless for green tea. Some unknown components were found in the extracts at 100 ${^{\circ}C}$. When the green tea was filtered and re-extracted with new fresh water, still some flavonoids were extracted. However, the contents of flavonoids in the third extract were found negligible. The flavonoid extraction rate of green tea powders was higher than that of green tea leaves, but flavonoid decomposition of green tea powders was also faster than that of green tea leaves. The traditional way of drinking green tea was found appropriate in view of flavonoids intake.

• Bulletin of the Korean Chemical Society
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• 제34권9호
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• pp.2760-2764
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• 2013

Geniposide is a biologically active ingredient of gardenia fruit. A liquid chromatography-tandem mass spectrometric method was developed and validated for the determination of geniposide in rat plasma. The plasma samples were pretreated by solid-phase extraction and introduced into a BDS Hypersil $C_{18}$ column ($50{\times}2.1mm$, $5{\mu}m$) for chromatographic separation. The mobile phase consisted of 0.1% formic acid and 0.1% formic acid in acetonitrile, and gradient elution was performed at a flow rate of 0.25 mL/min. For mass spectrometric detection, multiple reaction monitoring was performed via an electrospray ionization source in positive mode. The calibration curve for geniposide was linear ($r^2=0.997$) in the concentration range of $0.005-1{\mu}g/mL$. The intra- and inter-day accuracies and precisions fulfilled the required criteria (${\pm}15%$). The developed method was subsequently used for pharmacokinetic analysis of geniposide after oral administration to rats at a dose of 50 mg/kg. The mean maximum plasma concentration of geniposide was $0.68{\pm}0.29{\mu}g/mL$ at $0.44{\pm}0.13h$, and the mean area under the plasma concentration versus time curve was $1.46{\mu}g{\cdot}h/mL$.