비임상시험관리기준에서 독성시험 전 분석법 밸리데이션은 농도 설정 및 시료 제조 측면에서 중요하다. 시험기관에서는 의뢰받은 시험물질 2종에 대한 밸리데이션을 고성능액체크로마토그래피를 이용 하여 수행한 결과 특이성, 시스템 적합성, 직선성, 일내 재현성, 균질성, 안정성, 농도분석, 품질관리를 판정 기준에 만족하는 분석방법을 확립 및 검증하였다. 하지만 의뢰기관의 시험성적서상 표준물질 함량은 시험 기관의 결과보다 1.34배, 1.17배 높은 결과로 나타나 비임상시험관리기준에 적합한 분석법 밸리데이션을 통한 결과 도출이 신뢰성과 안정성 확보 측면에서 중요함을 확인하였다.

본 연구는 불법적으로 식품에 사용될 수 있는 부정물질 11종에 대한 안전관리 강화를 위해 정량 및 정성 분석이 가능한 HPLC-DAD와 LC-MS/MS를 검증하기 위해 수행 되었다. 확립된 시험법은 AOAC 가이드라인에 따라 직선성, 정밀성, 정량한계 및 회수율 등을 통해 유효성을 확인 하였다. 본 실험에서 정량한계를 포함하여 검량선을 작성 하였고, 모두 0.99 이상의 직선성을 확인하였다. 또한 정확성은 LC (90.0-106%), LC-MS/MS (83.0-114%) 이고, 정 밀도는10% 이하로 재현성이 우수하였다. 확립된 시험법은 식품 중 부정물질 안전관리 및 모니터링에 활용될 것으로 사료된다.

본 연구는 조제유류 중「식품의 기준 및 성분규격」에 기준 규격이 설정되어 있지 않으나 제품에 함유되어 있는 베타카로틴에 대해 분석법을 마련하고자 수행하였다. 조제유류에 함유된 베타카로틴 함량 분석을 위해 HPLC를 이용한 분석법을 확립하고 시중에 유통 중인 제품을 대상으로 적용성을 검토하였다. 베타카로틴 표준품을 이용하여 HPLC를 이용한 기기분석조건을 확립하고 시료 중의 베타카로틴을 추출하여 분석하였다. 분석법 검증은 특이성, 직선성, 검출한계 및 정량한계, 정확성, 정밀성에 대해 수행되었다. 0.125~2 mg/L의 농도범위에서 R2=0.999 이상의 우수한 직선성을 확인할 수 있었으며, LOD와 LOQ는 각각 0.1, 0.2 mg/L였다. 표준물질 첨가법을 이용하여 정 확성을 검토하였으며, 81~120%의 회수율을 확인할 수 있었다. 정밀성을 검토한 결과 시료 채취량에 따른 반복성은 RSD값이 2.1~4.9%, 실험실간 교차검증을 통한 실험일자에 따른 재현성은 4.0 RSD(%)로 확인되었다. 본 연구에서 확립된 분석법을 적용하여 조제유류 13건, 성장기용 조제식 7건 등 국내 유통 중인 제품 20건에 대해 적용성 검토를 실시한 결과 전체 시료에서 분석이 용이하였으며, 모두 표시기준에 적합함을 확인하였다.

A study of the tissue depletion of florfenicol (FFC) administered orally to pigs at a dose of 0.05 kg/ton feed for 7 days was performed. Sixteen healthy cross swine were administered with FFC. Four treated animals were arbitrarily selected to be sacrificed 1, 3 and 5 days after the end of treatment. FFC residue concentrations in muscle, liver, kidney, and fat were determined using high-performance liquid chromatography (HPLC) with ultraviolet photometric detector at 230 nm. The correlation coefficient (R2) of the calibration curve for florfenicol amine (FFCa) was > 0.997 and the limits of detection and quantification were 0.012 and 0.040 μg/mL, respectively. Recovery rates in swine edible tissues ranged from 79.1 to 93.5%. In the FFC-treated group, FFC residues at 3 days post-treatment were below the maximum residue limits (MRLs) in muscle, kidney and fat, and those at 5 days post-administration were below the MRLs in all edible tissues. These results suggest that the withdrawal period of FFC after the drug treatment might be 5 days, which is a sufficient amount of time for reduction of the FFC residues below the MRLs in all edible tissues.

5-fluorouracil (5-FU) is a pyrimidine analog which can work as antineoplastic antimetabolite by blocking thymidylate synthetase conversion of deoxyuridylic acid to thymidylic acid in DNA synthesis. This study is aimed to know the anticancer effect of 5-FU on the expressions of important signaling proteins in KB cells through immunoprecipitation high performance liquid chromatography (IP-HPLC). KB cells were treated with 5 μM 5-FU and cultured for 12, 24, 48, 72, and 96 hours, and followed by IP-HPLC analysis using 32 antisera. 5-FU suppressed the proliferation of KB cells by decreases in the expressions of proliferation-related proteins, Ki-67, PCNA, CDK4, and MPM2 to 82.6%, 92.4%, 95.2%, and 95.9%, respectively, but increases of antiproliferation-related proteins, p16 and p21 to 106.7% and 125.5%, respectively, during 96 hours of experiment. This proliferation reduction was also negatively regulated by cMyc/MAX/MAD network signaling. The cellular protection and survival were consistently arrested by 5-FU treatment in KB cells. The expressions of NFkB, MDR, p-mTOR, and TNFα were decreased to 95.1%, 92.8%, 93.4%, and 90.3% in 48-72 hours, respectively, while cellular stress was increased by upregulation of p38 to 111.3% in 48 hours. And the expressions of pAKT1/2/3, hTERT, and AMPK were also decreased to 93.3%, 97.4%, and 89.3% in 24-48 hours, respectively, while the cellular transformation might be undergone by upregulation of TGF-β1 to 117% until 96 hours. Particularly, 5-FU treatment greatly induced the cellular apoptosis in KB cells by increased expressions of PARP, cPARP, caspase 9, c-caspase 9, caspase 8, and caspase 3 in the lack of p53/BAX and FASL/FAS signaling. The expressions of PARP and c-PARP were increased maximum to 119.2% in 24 hours, and followed by increases of caspase 9, c-caspase 9, caspase 8, and caspase 3 to 111.2%, 125.9%, 108.6%, and 116.3% in 72-96 hours. Therefore, it is presumed that 5-FU induced cellular apoptosis in KB cells may be derived from the overexpression of PARP due to the increased DNA defect caused by 5-FU, which can lead to ATP depletion and subsequent cellular apoptosis.

The native human saliva obtained through the centrifugation of whole saliva showed characteristic salivary protein complex (SPC) peaks in gel filtration high performance liquid chromatography (HPLC) using Superose 12 column1,2). In the previous study the SPC peaks in chromatography were explored to know their composition and functions by different detection methods, but still the nature of SPCs was not clearly elucidated so far. In this study the SPC peaks were examined by direct antibody interaction in order to target different antimicrobial and protective proteins distributed in the SPCs via gel filtration HPLC. As the SPC peak shape and migration speed can be changed by antibody binding to specific proteins of SPC, it was found that mucin1 is evenly distribution in all SPCs, while PRPs are more abundant in the late dominant SPC than the early dominant SPC and also in the intermediated SPCs. Most of antimicrobial proteins including lysozyme, LL-37, lactoferrin, β-defensin-1, -2, -3, IgA, mucocidin, and α1-antitrypsin were more abundant in the late dominant SPC than the early dominant SPC, while histatin showed relatively even distribution in all SPCs. Therefore, it was presumed that the late dominant SPC containing abundant antimicrobial and protective proteins could be applied as a biomarker to measure the defensive potential of whole saliva in oral diseases.

In this study, we developed and validated microanalysis methods for the determination of linear alkylbenzenesulfonate (LAS), sodium lauryl sulfate (SLS), and alpha olefin sulfonate (AOS). The conditions for the analysis of the surfactants using HPLC with FLD, RID, and ELSD detectors were investigated. The methods were validated by determining the linearity, limits of detection (LODs), limits of quantification (LOQs), recovery, precision, and accuracy. LAS analysis by FLD revealed calibration curves that were linear in the range of 10-200 mg/L for an LAS mixture. The calibration curves for C10-C13 had correlation coefficients of 0.995, 0.997, 0.996, and 0.997, respectively. SLS analysis using RID generated a linear calibration curve in the range of 10-300 mg/L. The calibration curve for SLS C12 had a correlation coefficient of 0.9994. AOS analysis using ELSD resulted in a correlation coefficient of 0.9940. For LAS, the LODs and LOQs were 0.09-0.56 and 0.30-1.87 mg/L, respectively. For SLS C12, the LOD and LOQ were 0.07 and 2.33 mg/L, respectively. For AOS C14, the LOD and LOQ were 16.55 and 21.83 mg/L, respectively. The recoveries were 97.17-98.84% for LAS C10-C14, 97.94% for SLS C12, and 96.11% for AOS C14. The established methods provide acceptable precision and accuracy. Our methods could be useful for the detection of anionic surfactants in dishwashing detergents.

Immunoprecipitation-based high performance liquid chromatography (IP-HPLC) is a type of modified enzyme-linked immunosorbent assay (ELISA) that uses protein A/G (or antibody)-conjugated beads instead of the antibody-conjugated wells used in ELISA. In order to determine the fidelity of IP-HPLC, the author used 83 antisera to identify protein expression changes caused by cisplatin treatment in KB human oral cancer cells. KB cells were cultured for 12 or 24 hours with 10 ug/mL cisplatin. The results obtained by IP-HPLC were comparable with published cisplatin data, although ELISA was not conducted in the present study. Cisplatin dominantly reduced the levels of proteins associated with cell proliferation, transcription factors, growth factors, cytoskeletal proteins, and cellular differentiating factors, but on the other hand, apoptosis-related factors, oncogenes, and protective proteins were usually up-regulated, presumably to address cisplatin-induced DNA damage. In particular, cisplatin directly inactivated genomic DNA by down-regulating histone H1 and demethylase and by up-regulating deacetylase. Cisplatin also rapidly induced p53 overexpression and mitochondria-mediated endogenous apoptosis occurred after 12 hours of cisplatin treatment, although this was almost completely replaced by FASL/FAS-mediated exogenous apoptosis after 24 hours. This preliminary study was conducted to investigate the anticancer effect of cisplatin on the KB human oral cancer cells and to determine the fidelity of IP-HPLC data. It was concluded that IP-HPLC is useful for identifying profile changes of genome wide essential proteins and signaling changes of major molecular pathways.

Salivary proteins include numerous functional proteins which play important roles not only for the food-intake but also for the protective and defensive mechanisms. In the present study the compositions of salivary proteins were analyzed by different methods, including electrophoresis and high performance liquid chromatography (HPLC). In hydrophobic protein HPLC analysis the parotid saliva gradually produced macromolecular complexes when agitated in refrigerator until 30 minutes. These salivary protein complexes were digested by neuraminidase, and then migrated more rapidly in native tris glycine gel than the control. Therefore, it was assumed that the glycosylated proteins of parotid saliva became gradually aggregated to form salivary protein complexes similar to those of whole saliva. The salivary protein complexes were easily degenerated in different experimental buffers, i.e., SDS buffer, tris glycine buffer, methanol, etc., and resulted non-specific patterns in electrophoresis and HPLC. Therefore, it was presumed that the salivary protein complexes was made by the hydrophobic interaction as well as electrostatic attraction between salivary proteins. These data indicated that to know the real pattern of salivary protein complexes in vivo the whole saliva should be analyzed by HPLC using non-adhering column with isoelectric buffer. Consequently, the whole saliva was analyzed by HPLC using reverse phase SuperoseTM column with 20 mM potassium phosphate buffer, and two prominent peaks of salivary protein complexes were consistently found in every people. These salivary protein complex peaks were relatively stable up to 6 hours after saliva collection when the whole saliva was kept in refrigerator during experiment. Therefore, it is suggested that the salivary protein complex patterns are characteristic macromolecular structures of whole saliva, which are also applicable as a diagnostic point in different saliva-associated diseases

The molecular mechanisms of the carcinogenesis of oral squamous cell carcinomas (OSCCs) are highly variable and result in different features of tumor progression, i.e., local tissue destruction and metastasis to regional lymph nodes. A case of OSCC arising from proliferative verrucous leukoplakia (PVL) was analyzed for its protein expression profile by immunoprecipitation (IP) – high performance liquid chromatography (IP-HPLC) by using 72 antisera and comparing results with those of KB cells. OSCC arising from PVL showed stronger expressions of proteins associated with cell proliferation (MPM2, PCNA, eiF5A, DHS, DOHH), cell survival (pAKT, MDM2, survivin), matrix proteolysis (elaffin), tumor suppression (p16, p21, PTCH1), the WNT/β-catenin pathway (SHH, WNT1, APC, β-catenin, snail), proinflammation (TNFα), angiogenesis (HIF, CMG2, vWF), and cellular protection (HSP-70, FAK, caveolin) and of oncoproteins (STAT3, 14-3-3, K-RAS, PUMA, PIM1) and growth factors (EGFR, bFGF) than KB cells. On the other hand, KB cells showed stronger expressions of proteins associated with apoptosis (caspase-3, caspase-8, caspase-9, PARP, FAS, FASL, TGase-1, BCL2, BAD, BID, BAK, FLIP), matrix proteolysis (MMP-2, MMP-9), transcription signaling (NFkB, p38, E2F-1, HO-1), and tumor suppression (p53, RB1, PTEN) and of oncoproteins (DMBT1, CEA) and growth factor (TGF-β1, c-erbB2, VEGF) than OSCC arising from PVL. These data indicate the cells of OSCC arising from PVL are more resistant and more robust than KB cells. Furthermore, they suggest the oncogenic signalings of OSCC arising from PVL play important roles in the aggressive growth and rapid tumor metastasis to regional lymph nodes

영지버섯의 유용한 생리활성을 나타내는 물질은 주로 버섯의 자실체로부터 분리ㆍ동정되었 으며, 이로 인하여 현재까지 다양한 영지버섯의 자실체를 대상으로 그 분석이 수행되고 있다. 그러나 최근 버섯의 균사체에서도 다양한 생리활성을 지니는 물질이 분리ㆍ동정되고 있으며, 이에 따라 버섯의 균사체를 대상으로 하는 생화학적 분석방법의 표준화 등이 요구되고 있는 실정이다. 또한 균사체는 자실체와 달리 배양기간이 짧고, 비용이 적게 소요되어 효율적인 분 석이 가능하다. 그러나 버섯의 균사체는 고분자인 다당류의 함량이 자실체보다 많아 역상 (Reverse phase) 컬럼을 주로 이용하는 HPLC (High Performance Liquid Chromatography) 의 화학적 분석이 용이하지 않다. 이에 본 연구에서는 분석이 어려웠던 균사체 세포 내의 대 사물질 프로파일을 확보하기 위해 부탄올을 이용한 용매 계통 분획으로 고분자 다당류를 제 거하고, HPLC 이동상의 선형 구배 (Linear gradient) 조건을 확립하여 저분자 물질들에 대한 분석 결과를 관찰하였다. 본 연구 결과는 향후 버섯 균사체에 대한 대사체 분석 기술의 기반 이 될 것으로 사료된다.

본 논문에서는 축산물 중 닭고기 내 마크로라이드계(MLs) 항생물질(spiramycin, josamycin, tilmicosin, 그리고tylosin) 4종을 분석하기 위하여, 액-액 추출 과정을 거쳐서 PDA 검출기가 장착된 액체크로마토그래피를 이용하여MLs를 효율적으로 동시분석하는 방법을 확립하였다. 컬럼은 Unison UK-C18 (150 mm × 3 mm id, 3 μm), 이동상 용매는 0.1% TFA와 0.1% TFA in ACN로 기울기 용리를사용하였으며, 유속은 0.7 mL/min, 그리고, 주입량은 10 μL로 설정하여 분석하였다. 확립된 분석조건으로, 표준검정곡선은 50-1000 μg/kg의 농도범위에서 상관계수가 0.9975이상의 양호한 직선성을 나타내었으며, 회수율은 저, 중,고농도로 첨가하여 분석한 결과, 80.4-99.1%를 나타내었다. 검출한계는 8.8-19.6 μg/kg이었고, 정량한계는 26.6-59.4μg/kg이었으며, 일내(intra-day)와 일간(inter-day) 정밀도(CV%)는 0.9-13.2%, 2.4-13.1%이었다. 따라서, 확립된 분석방법은 축산물 중 닭고기 시료 내 MLs를 효과적으로분석하는데 이용될 수 있을 것으로 사료된다.

As a preceding study for investigating the influence of sound wave stimulus on Arabidopsis thaliana metabolomics, the polar secondary metabolomes of the plant were determined using high performance liquid chromatography coupled with tandem mass spectrometry. A total of 10 polar secondary metabolomes were characterized and quantified. Among them, 4 metabolomes, p-coumaroylagmatine isomer (7 and 8), p-coumaroylagmatine isomer (9 and 10) were identified in the plant for the first time. The validation was conducted in terms of linearity, recovery, precision, limit of detection (LOD) and limit of quantification (LOQ). The validated method was applied to the simultaneous quantification of the 10 polar secondary metabolomes.

Deoxynivalenol (DON) and zearalenone (ZEN) are mainly contaminated mycotoxins in feeds. The study was carried out to analyze and survey the contamination of DON and ZEN in one hundred thirteen samples of feeds. After cleaning all samples with immunoaffinity column, the mycotoxins were analysed by using high performance liquid chromatography/fluorescence with diode array detector (HPLC /FLD with DAD). The average recoveries of DON were 88.76 and 95.40% at the levels of 200 and 1,000 μg/kg and 87.09 and 98.40% of ZEN were recovered at the levels of 100 and 500 μg/kg, respectively. The limit of detection (LOD) were 6.0 and 3.0 μg/kg for DON and ZEN, respectively. The average concentrations of DON were 372.1, 324.0 and 990.9 μg/kg in chicken, pig and cattle feed, respectively. Those of ZEN were 76.1, 43.7 and 196.2 μg/kg for them, individually.

A method was established for the simultaneous determinatioin of sugar alcohols, erythritol, xylitol,sorbitol, inositol, mannitol, maltitiol, lactitol and isomalt by High Performance Liquid Chromatography (HPLC). The sugar alcohols were converted into strong ultraviolet (UV)-absorbing derivatives with p-nitrobenzoyl chloride (PNBC). HPLC was performed on Imtakt Unison US-C18 column, using acetonitrile : water (77:23) as a mobile phase and UV detection (260 nm). The calibration curves for all sugar alcohols tested were linear in the 10~200 mg/L range. The average recoveries of the sugar alcohols from three confectioneries spiked at 100 ppm of eight sugar alcohol standards ranged from 81.2 to 123.1% with relative standard deviations ranging fromo 0.2 to 4.9%. The limits of detection (LODs) were 0.5~8 μg/L and the limits of quantification (LOQs) were 2~17 μg/L. Reproducibility of 8 sugar alcohols was 0.28~1.97 %RSD. The results of the analysis of confectioneries showed that 89 samples of 130 were detected and the sugar alcohols content of samples investigated varied between 0.4 and 693.7 g/kg. A method for the simultaneous determination of eight sugar alcohols will be used as basic data for control of sugar alcohols in confectioneries, and qualilty control in food manufacturing.

목적: In vitro 조건 하에서 재질이 다른 실리콘 하이드로겔 렌즈에 결합되는 눈물의 지방성분인 올레산(oleic acid), 올레산에스테르(oleic acid methyl ester) 및 콜레스테롤(cholesterol)을 각각 정량하여 실리콘 하이드로겔렌즈에 부착되는 지방침전물을 비교하였다. 방법: Lotrafilcon A, lotrafilcon B, galyficon A 및 balafilcon A 재질의 실리콘 하이드로겔 렌즈와polymacon 렌즈 및 RGP 렌즈를 올레산, 올레산에스테르 및 콜레스테롤을 동일한 양 포함하는 식염수 용액에 담가 37℃에서 흔들어주면서 24시간 동안 오염시킨 후, methanol과 chloroform을 1:1(v/v) 비율로 포함하는 유기용매를 이용하여 4시간 동안 렌즈에 부착된 지방을 추출하였다. 추출된 지방은 C-18 column을 사용하여 HPLC(high performance liquid chromatography)를 수행하여 분리하고 정량하였다. 결과: 실리콘 하이드로겔렌즈에 부착된 지방의 총량은 재질의 종류에 따라 48~67 ㎍/lens, polymacon 렌즈와 RGP 렌즈에서는 각각 18 ㎍/lens, 177 ㎍/lens로 측정되어, 실리콘 하이드로겔렌즈의 지방 부착량은 polymacon 소프트렌즈의 300~372%, RGP 렌즈의 30.5~37.8%로 나타났다. 지방침전물은 galyfilcon A 재질에 가장 많이 부착되었으며(p

This study was conducted to establish a method to analyze azodicarbonamide (ADA) in wheat flour. A new method using high performance liquid chromatography (HPLC) was developed for the determination of ADA in wheat flour. The recovery rate was 91.93~97.54%. The limit of detection for ADA was 0.02 mg/kg and the limit of quantification was 0.05 mg/kg. The monitoring results for ADA contents using the established methods showed that it was detected as the low value of 0.95 mg/kg in one of 51 flour samples (detection rate : 2%), but not detected in 59 bakery samples. The detected ADA level was suitable to its usage standard, compared to the standard (45 mg/kg). Although the detection rate was very low, the established analytical method of ADA will contribute to the management of ADA in processed foods such as wheat flour and bakery.

As the human mixed saliva plays important roles for the protection, regeneration, immunity, and molecular transfer/ signaling in the oral and gastro-intestinal mucosa, the salivary contents have great implications for the general health of human body. Nevertheless, the analysis method of human saliva has not been well developed up to date, because the proteins of mixed saliva are rapidly interacted with each other and easily degraded by proteolytic enzymes and microorganisms. This study aims to develop an immunoprecipitation-based high performance liquid chromatography (IP-HPLC) for the analysis of human mixed saliva. The representative IP-HPLC analyses were performed to compare among different subjects in variable general conditions. Compared to the normal control the subjects suffered from bacterial infections of gastro-intestinal enteritis, chronic periodontitis, and acute necrotizing gingivo-stomatitis showed dramatic increase of LL-37 level depending on the severity of diseases, while the subject suffered from Herpes stomatitis, a viral infection showed great increase of β-defensin 2. These data indicate that LL-37 in human mixed saliva is more responsible to the bacterial infections of gastro-intestinal enteritis, chronic periodontitis, and acute necrotizing gingivo-stomatitis, while β-defensin 2 is more responsible to the viral infection of Herpes stomatitis. This study also suggeststhat the IP-HPLC be easily applicable to the wide range of biological samples for the quantitative analysis of an objective protein.

HPLC에 의한 주요 aflatoxins(afatoxin B₁, B₂, G₁ 및 G₂)의 동시 분석에서 postcolumn 유도체화법을 시도하였다. Electrochemical cell(Kobra-cell)을 사용한 postcolumn 유도체화법은 기존의 precolumn 유도체화법보다 분석시간을 단축하였으며(약 1/2 단축), 더 안전하고, 향상된 분석능을 보였다. Aflatoxin B₁과 G₁의 경우 10~100 ppb에서, 그리고 B₂와 G₂의 경우 3~30 ppb에서 직선성을 나타내었다. Aflatoxin B₁과 G₁은 각각 88.9% 및 100.5%로 양호환 회수육을 보였다. Aflatoxin B₂와 G₂의 경우 분리도는 우수하였으나 회수율에 있어서 변이가 크게 나타났다.