A liquid chromatographic-tandem mass spectrometric (LC-MS/MS) multi-residue method for simultaneous quantification and identification of 37 anthelmintic veterinary drug residues (including benzimidazoles, macrocyclic lactones, and flukicides, levamisole, pyrantel and niclosamide) in milk has been developed and validated. For sample preparation, we used a simple modification of the QuEChERS (quick, easy, cheap, effective, rugged and safe) method, which was initially developed for analysis of pesticide residues. Anthelmintic residues were extracted into acetonitrile:methanol (9:1, v/v) using sodium chloride to induce liquid-liquid partitioning followed by dispersive solid phase extraction for cleanup. The extract was concentrated into dimethyl sulphoxide, which was used as a keeper to ensure that analytes remain in solution. Using rapid polarity switching in electrospray ionization, a single injection was capable of detecting both positively and negatively charged ions within a 15 min run time. The Limit of detection (LOD) and the Limit of quantification (LOQ) method ranged from 0.1 ng/g to 4.4 ng/g and from 0.3 ng/g to 14.6 ng/g, respectively. Validation of the developed method was based on international guidelines. Average recoveries ranged from 70% to 120%, except for 54.7% at 0.5× MRL (rafoxanide) and 69.0% at 0.5× MRL (closantel). The coefficient of variation for the described method was less than 15% over the range of concentrations studied. The result of the method was verified successfully by participation in a proficiency study for analysis of anthelmintic drugs.

A confirmatory method based on liquid chromatography with tandem mass spectrometry was developed for determination of 12 aminoglycosides in milk. Extraction of aminoglycosides from milk was performed using by liquid extraction using a 10 mM phosphate buffer containing 2% (w/v) trichloroacetic acid, followed by performance of a solid-phase clean-up procedure on a hydrophilic-lipophilic balance solid-phase extraction (HLB SPE). Ion-pair chromatography, using a mixture of 20 mM heptafluorobutyric acid (HFBA) in water and acetonitrile as the mobile phase, was used for retention of aminoglycosides on a reversed-phase C18 column. Mass spectral acquisition was performed in the multiple reaction monitoring mode, selecting two precursor ion>product ion transitions for each target compound. Satisfied recoveries (70.1～109.6%) of all aminoglycosides were demonstrated in spiked milk at three levels from 50 ng/g to 200 ng/g. The coefficients of variation ranged from 3.2% to 14.0%. The limits of quantitation (LOQs) for aminoglycosides ranged from 2.5 ng/g to 40.3 ng/g.

A rapid, simple and reliable LC-MS/MS method, which can be used on a routine basis, was developed for the simultaneous detection of 8 penicillin antibiotics (amoxicillin, ampicillin, penicillin-G, penicillin-V, oxacillin, cloxacillin, nafcillin and dicloxacillin) in swine muscle and kidney. The antibiotics were extracted from samples with water and methanol. The extract was centrifuged, filtered and analyzed by liquid chromatography coupled with a tandem mass spectrometer (LC-MS/MS), using a C18 reversed phase column with water/acetonitrile gradient containing 0.05 % formic acid. Mass spectral acquisition was achieved in an electrospray positive ion mode by applying multiple reaction monitoring (MRM) of 2 fragment ion transitions to provide a high degree of sensitivity and specificity. The ion rations were consistent and could be used for confirmation of identity of the penicillin antibiotics. Recoveries of eight penicillins at three fortification levels (10, 20 and 40㎍/㎏) ranged from 79.8 to 102.4% and 72.8 to 103.4% in swine muscle and kidney, respectively. The coefficient of variation was than 9% in all samples. The estimated limits of quantification (LOQs) ranged from 1.0 to 3.2㎍/㎏ in swine muscle and kidney, respectively. The LOQs of this method are below the MRLs of penicillin antibiotics in animal tissues established in Korea and other countries.

인간성장호르몬(human growth hormone; hGH)은 뇌하수체 전엽에서 분비되는 호르몬 의 일종으로 단백질 합성을 촉진하고 에너지를 생산해 지방을 분해하며 뼈를 포함한 체 내의 거의 모든 조직의 성장을 자극한다. DNA 재조합 기술이 발달함에 따라 hGH는 신 체의 성장, 골밀도 향상, 체지방 감소, 세포의 재생, 근육량 증가 등에 효과를 보이는 의 약품으로 사용되고 있다. 본 연구에서는 시료 중 함유된 hGH의 효율적인 분리,정제를 위 하여 immunoaffinity chromatography system을 개발하여 hGH를 분리정제하고 그 활성도 를 측정하고자 하였다. 이를 위하여, 단크론성 항체를 생산하는 hybridoma cell line중 hGH에 가장 강한 친화력이 있는 항체를 분비하는 hGH-K-24 세포주를 선별하여 Balb/C 마우스의 복강에 주입하여 복수를 생산, 채취하였고, Protein G를 이용하여 항체를 분리 정제 하였다. 정제한 항체를 CNBr-Sepharose 4B와 결합시켜 immunoaffinity chromatography column을 제작하였고. hGH를 분비,생성하는 CHO세포주를 serum이 없는 상태에 서 10일 동안 배양한 다음 배지를 회수하여 hGH를 immunoaffinity chromatography system으로 정제하였으며, 순수정제도를 높이기 위하여 superdex G-200으로 gel filtration chromatography 하여 target 단백질인 hGH만을 분리하는 일련의 과정을 확립하였다. 또 한, hGH의 역가를 알아보기 위하여 NB2-11 cell을 이용하여 MTT assay를 통한 활성도 를 살펴보았다.

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.

The aim of this study was to investigate what protein(s) of porcine epididymal fluid (pEF) are able to enhance the nuclear maturation of porcine germinal vesicle (GV) oocytes in vitro. Proteins of pEF were fractionated by affinity, ion exchange, and gel filtration chromatography. Porcine cumulus-oocytes complexes (COC) from follicles were cultured in tissue culture medium (TCM 199) containing various fractions obtained by chromatography. Porcine COCs were also cultured in TCM 199 containing various meiosis inhibitors and pEF. After 24 or 48 h culture, oocytes were examined for evidence of GV breakdown, metaphase I, anaphase-telophase I, and metaphase II. When porcine COCs were cultured in the medium with meiosis inhibitor such as, dibutyryl cAMP (dbcAMP) and forskolin (Fo), more than 80% of oocytes were unable to resume meiosis. However, porcine COCs supplemented with pEF were able to overcome the inhibitory effect of dbcAMP and Fo. Maturation rate of oocytes was significantly (p<0.05) increased in the media supplemented with cationic protein(s) during in vitro maturation than in those with anionic protein(s) (44.1% vs 20.0%). When oocytes were cultured in the TCM 199 with fractions obtained by gel filtration, the maturation rate of oocytes was significantly (p<0.05) higher in fraction 11 containing 18 kDa than other fractions. The present study suggests that 1) dbcAMP and Fo prevent the spontaneous maturation of oocyte after isolation from follicles, and that pEF contain a substance(s) that improves meiosis resumption in vitro of porcine COCs, 2) cationic 18 kDa protein(s) are responsible for promotion of MⅡ stage.

Tomato fruits(Lycoperisicon esculentum) synthesize the glycoalkaloids dehydrotomatine and α-tomatine, possibly as defense against bacteria, fungi and insects. We developed a new effective method to prepare and purify dehydrotomatine and α-tomatine that exists in tomato fruits using alumina column chromatography and high performance liquid chromatography (HPLC). The tomato glycoalkaloids(TGA) in tomato was extracted with 2% acetic acid, and then precipitated with ammonium hydroxide(pH=10.5). The dry precipitate substance was applied on alumina column, and then fractionated with water saturated n-butylalcohol. The TGA(Fr. No. 26~36) were collected and dried under reduced pressure. The TGA was performed on a reverse phase HPLC(Inertsil ODS-2, 5 ㎛), eluted with acetonitrile/20mM KH2PO4(24:76, v/v) at 208 ㎚. Two peaks were detected on HPLC, and individual peak was collected by repeating HPLC. Furthermore, to confirm the identity dehydrotomatine and α-tomatine, each peak isolated was hydrolyzed with 1N HCl into sugar and aglycone tomatidine. The sugars were converted to trimethylsilyl ester derivatives. The nature and molar ratios of sugars were identified by gas-liquid chromatography(GLC) and the aglycone by high-performance liquid chromatography(HPLC). The first peak (Rt=17.5 min) eluted from HPLC was identified as dehydrotomatine, and second peak(Rt=21.0 min) was as α-tomatine. This technique has been used effectively to prepare and isolate dehydrotomatine and α-tomatine from tomato fruits.

LC-DAD-ESI/MS를 이용하여 국내 자색벼 품종에 대해 개별 안토시아닌 조성 및 함량을 평가한 결과는 다음과 같다. 1. 자색벼 품종에서 분리된 모든 개별 안토시아닌의 화학구조는 MS fragment 패턴을 확인하여 cyanidin을 base로 한 unknown 화합물 1종을 포함, cyanidin 3,5-diglucoside, cyanidin 3-glucoside, peonidin 3-glucoside의 총 4가지 개별성분이 분리 및 동정되었다. 2. Cyanidin 3-glucoside 및 peonidin 3-glucoside이 주요성분으로 cyanidin 3-glucoside의 경우 90% 이상의 가장 높은 함량 비중을 나타냈으며, 개별성분별 평균 함량은 cyanidin 3-glucoside > peonidin 3-glucoside > cyanidin 3,5-diglucoside > unknown(cyanidin based)의 순으로 나타났다. 3. 흑진주벼의 총 안토시아닌 함량은 408 mg/100 g으로 흑남벼보다 약 6배 정도 높은 함량을 나타내었다.

A simple, selective and sensitive procedure for the confirmation of 14 sulfonamide antibacterials in milk was developed. The milk samples were homogenized, extracted and deproteinized by acetonitrile and defatted by n-hexane. Analysis was performed by liquid chromatography with tandem mass spectrometry (LC-MS/MS) in positive mode for all 14 analytes. Mass spectral acquisition was performed in the multi reaction monitoring mode (MRM), selecting two structurally significant transitions per compound. The calibrations were performed in sample matrixes and the interference effect of sample matrixes on the ionization was effectively eliminated. Good linear relationship (R2=0.992～ 0.999) were observed at 6 concentrations of 2.5～100 ng/g. Satisfied recoveries (86.3～110.2%) of all sulfonamides were demonstrated in spiked milk at three levels from 5 to 20 ng/g. The limits of quantitation (LOQs) for sulfonamides ranged from 0.25～2.1 ng/g.

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.

The pomegranate (Punica granatum), especially its fruit, possesses a vast ethnomedical history and represents a phytochemical reservoir of heuristic medical value. The tree and fruit can be divided into several anatomical compartments, and the fruit juice, peel and oil are known to be weakly estrogenic and heuristically of interest for treatment of menopausal symptoms and sequellae. In this study, analysis of estrogen in pomegranate extract was carried out with LC/MS/MS. Three batches of pomegranate extract samples were used to analysis the target compounds (estrogen). The contents of estrogen derivatives in the samples were 38.6 ppb of estriol, 83.5 ppb of estrone,and 10.9 ppb of estradiol. This result suggests that the pomegranate extract can used for treatment of menopause symptoms in the woman.

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.

In this study, we have developed a fluorescence chromatographic assay for the quantification of total cholesterol in serum, which is a well-known risk predictor for cardiovascular diseases. The new assay system consists of a chromatographic strip in a cartridge, enzyme buffer containing cholesterol esterase, cholesterol oxidase, horseradish peroxidase, and color developer AEC, and a laser fluorescence scanner. The correlation coefficient (r) between cholesterol concentration and relative fluorescence units was 0.968 in the new assay, showing a reliable linearity through the tested range of cholesterol. Recovery test and comparability with a Hitachi 747 instrument showed 106.5-94% and r = 0.939 (p<0.001), respectively. The new assay system for cholesterol was developed as a pre-POCT platform conducted in clinics since it is fast (8 min) and uses a small volume of sample (5 μl), and it may be applied for on-site diagnostics to replace expensive automated biochemical analyzer.

실리카 입자를 기공 형성제로 사용하여 물리적 강도와 단백질 결합용량이 높은 다공성 키토산 및 키틴 친화 막을 제조하였다. 키토산 친화 막의 BSA 단백질 결합용량은 최대 21.8mg/mL이었으며, 키틴 친화 막의 lysozyme 효소 결합용량은 최대 26.1mg/mL이었다. 제조된 다공성 키토산 및 키틴 친화 막을 사용하여 단백질 용액의 loading 유량, loading 양 및 농도 변화에 따른 BSA와 lysozyme의 친화 막 여과 크로마토그래피 분리 실험을 수행하였다. 친화 막 여과 크로마토그래피 분리 실험을 통해 얻어진 loading/washing/elution의 단계로 구성된 일련의 크로마토그램으로부터 단백질 용출량과 결합수율을 구하였다. 키토산 및 키틴 친화 막에의 BSA 및 lysozyme 단백질의 결합량과 결합수율은 loading용액의 유량이 작을수록, 주입량 및 농도가 클수록 증가하였다. 이 결과로부터 실리카 입자를 기공 형성제로 사용하여 제조된 다공성 키토산 및 키틴 막은 단백질의 대규모 여과 크로마토그래피 분리를 위한 친화 막으로서 효과적인 활용이 기대된다.

본 논문에서는 식품 중 퀴놀론계(QNs) 합성항균제(marbofloxacin, norfloxacin, danofloxacin, ciprofloxacin, enrofloxacin, difloxacin, sarafloxacin, oxolinic acid 그리고 flumequine) 9종을 분석하기 위하여, 액-액 추출 과정을 거쳐서 형광검출기가 장착된 액체크로마토그래피를 이용하여 QNs를 효율적으로 동시분석하는 방법을 확립하였다. 컬럼은 Zorbax Eclipse XDB-C8(150 mm×4.6 mm, 5 μm), 이동상 용매는 200 mM ammonium acetate buffer (pH 4.5)와 ACN로 기울기 용리를 사용하였으며, 유속은 1.5 ml/min, 그리고, 주입량은 10 μl로 설정하여 분석하였다. 확립 된 분석조건으로, 표준검정 곡선은 10-500 μg/kg의 농도범위에서 상관계수가 0.9989 이상의 양호한 직선성을 나타내었으며, 회수율은 50, 100 그리고 500 μg/kg의 농도에서 89.6-106.5 %로, 향상된 추출효율을 나타내었다. 검출한계는 1-16 μg/kg이었고, 정량한계는 3-47 μg/kg이었으며, 일내(intra-day)와 일간(inter-day) 정밀도(CV%)는 0.2-15.0 %, 0.5-11.7 %이었다. 따라서, 확립된 분석방법은 광어 및 계란 중의 QNs을 효과적으로 분석하는데 이용될 수 있을 것으로 사료된다.