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Plant regeneration through the callus culture induced from bulb scales of an endangered species Lilium cernum Komarvo. (멸종위기종인 솔나리(Lilium cernum Komarvo.)의 인편 유래 캘러스 유도 및 기내 식물체 재분화)

  • Bae, Kee-Hwa;Yoon, Eui-Soo
    • Journal of Plant Biotechnology
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    • v.40 no.2
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    • pp.65-71
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    • 2013
  • Lilium cernum Komarvo. is an important endangered plant belonging to the family Liliaceae. A method was developed for the rapid micropropagation of L. cernum through plant regeneration from bulb scales explant-derived calli. The bulb scales segments were cultured on Murashige and Skoog (MS) medium supplemented with 0, 0.5, 1.0, $3.0mg{\cdot}L^{-1}$ kinetin and 0, 0.1, 0.5, $1.0mg{\cdot}L^{-1}$ NAA or 2,4-D for callus induction. In media with $0.5{\sim}3.0mg{\cdot}L^{-1}$ kinetin and $0.1{\sim}1.0mg{\cdot}L^{-1}$ NAA and 2,4-D, 95~100% of explants produced callus. They were then transferred to MS medium supplemented with various concentrations of NAA (0, 0.01, 0.05 and $1.0mg{\cdot}L^{-1}$) in combination with BA (0, 1.0 and $2.0mg{\cdot}L^{-1}$) for bulbet formation. Bulbet induction (78%), weight (468 mg) and size (15.5 mm) were obtained the highest on MS medium containing $2.0mg{\cdot}L^{-1}$ BA and $1.0mg{\cdot}L^{-1}$ NAA. In vitro frequency of plant regeneration was not significantly treated in strength of MS and sucrose concentration. Chlorophyll contents in 1/2MS with $50g{\cdot}L^{-1}$ sucrose treatments were higher than those in control and another treatment. This in vitro propagation protocol will be useful for conservation and mass propagation of this endangered plant.

Validation of the LC-MS/MS Method for Ginsenoside Rb1 Analysis in Human Plasma (LC-MS/MS를 이용한 인체 혈장에서 Ginsenoside Rb1의 분석법 검증)

  • Han, Song-Hee;Kim, Yunjeong;Jeon, Ji-Young;Hwang, Minho;Im, Yong-Jin;Lee, Sun Young;Chae, Soo-Wan;Kim, Min-Gul
    • Journal of the Korean Society of Food Science and Nutrition
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    • v.41 no.12
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    • pp.1753-1757
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    • 2012
  • A new liquid chromatographic tandem mass spectrometric (LC-MS/MS) assay for the quantification of ginsenoside Rb1 in human plasma was developed and validated. The separation was performed on a Agilent C18 column ($4.6mm{\times}150mm$, particle size 5 ${\mu}m$) with a gradient elution of 0.1% formic acid in water and 0.1% formic acid in methanol and a flow rate of 0.9 mL/min. The analyte was determined using electrospray positive ionization mass spectrometry in the multiple reaction monitoring (MRM) mode (m/z 1131.714${\rightarrow}$365.303). Human plasma samples were extracted with acetone : water (50:50) by the liquid-liquid extraction method. The method was linear over the dynamic range of 10~500 ng/mL with a correlation coefficient of r=0.9995. The intra-and inter-day precision over the concentration range of ginsenoside Rb1 was lower than 5.8% (correlation of variance, CV), and the accuracy was between 96.0~104.6%. This LC-MS/MS assay of ginsenoside Rb1 in human plasma is applicable for quantification in a pharmacokinetic study.

Analytical Characteristics of GC/MS and HPLC according to the Concentration Distribution of PAHs (PAHs 농도 분포에 따른 GC/MS와 HPLC의 분석특성에 관한 연구)

  • Hong, Jwa-Ryung;Choi, Kwang-Min
    • Journal of Korean Society of Occupational and Environmental Hygiene
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    • v.25 no.3
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    • pp.312-321
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    • 2015
  • Objectives: The purpose of this study was to determine the best method to analyze PAHs at extremely low concentrations. To this end, 16 PAHswere analyzed simultaneously by GC/MS, HPLC/FLD and HPLC/UVD, and the analytical characteristics of HPLC and GC/MS were compared. Methods: This study was conducted by GC/MS and HPLC/FLD/UVD, and evaluated linearity, precision and detection limit. Standard solutions were prepared for 21 samples in the range of $0.00001{\sim}1.0{\mu}g/mL$ and the samples were divided into four groups. All samples were made in three sets and analysis was replicated seven times. Results: Sixteen PAHs could be simultaneously separated by HPLC and GC/MS, and the adequate equipment was HPLC/FLD. The retention times by HPLC were shorter than GC/MS, and HPLC had better separation for most PAHs than GC/MS. The peaks of naphthalene and naphthalene-D8 partially overlapped for GC/MS. HPLC/FLD had a 20-2000 times lower limit of detection than GC/MS and UVD. However FLD was not adequate for analyzing acenaphthylene because it has too low a fluorescence quantum yield to be detected. The precision of HPLC/FLD/UVD and GC/MS showed less than 20% at $0.001{\mu}g/mL$ PAHs and when the concentration was higher, the coefficient of variation was decreased. HPLC/FLD was better for the overall detection of limits. Conclusions: The results indicate that the HPLC/FLD method has good linear range, precision and a detection of limits from $0.00001{\sim}0.0001{\mu}g/mL$ for all 16 PAHs. This study contributes to providing useful data for analysis technology and can be applied to occupational exposure measurement for PAHs in workplaces.

Validation of LC-MS/MS method for determination of ginsenoside Rg1 in human plasma (인체 혈장 중 Ginsenoside Rg1의 정량을 위한 LC-MS/MS 분석법 검증)

  • Kim, Yunjeong;Han, Song-Hee;Jeon, Ji-Young;Hwang, Min-Ho;Im, Yong-Jin;Lee, Sun Young;Chae, Soo-Wan;Kim, Min-Gul
    • Analytical Science and Technology
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    • v.26 no.4
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    • pp.221-227
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    • 2013
  • A sensitive and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed for the investigation of the ginsenoside Rg1 in human plasma. After addition of internal standard (digoxin), plasma was diluted with acetone and methanol (80:20), the supernatant was concentrated and analyzed by LC-MS/MS. The optimal chromatographic separation was achieved on an Agilent Eclipse XDB-C18 column ($4.6{\times}150mm$, $5{\mu}m$) with a mobile phase of 0.1% formic acid in water and 0.1% formic acid in methanol at a flow rate of 0.9 mL/min gradient mode. The standard calibration curve for ginsenoside Rg1 was linear ($r^2=0.9995$) over the concentration range 1~500 ng/mL in human plasma. The intra- and inter-day precision over the concentration range of ginsenoside Rg1 was lower than 7.53% (correlation of variance, CV), and accuracy exceeded 98.28%. This LC-MS/MS assay of ginsenoside Rg1 in human plasma is applicable for quantifying in the pharmacokinetic study.

Analysis of Pesticides in Herbal Medicine by QuEChERS and GC-MS/MS (QuEChERS 전처리와 GC-MS/MS를 활용한 생약의 잔류농약 분석 가능성 연구)

  • Seo, Go Eun;Kim, A Young;Pyo, Byoung Sik;Lee, Kyoung in
    • Korean Journal of Pharmacognosy
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    • v.51 no.3
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    • pp.207-216
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    • 2020
  • The analysis method for qualitative analysis of the screening method that can be performed prior to the quantitative analysis of individual pesticide was reviewed in order to meet the safety standards that are being strengthened in the field of pesticide residue testing of herbal medicines. Among the residual pesticides presented in the Korean Pharmacopoeia, 56 pesticides, excluding 15 pesticides that need to be individually analyzed, were selected for analysis using QuEChERS preprocessing and GC-MS/MS, which are used in the existing agricultural products field. For each pesticide, the detection limit level of 0.001-0.005 mg/kg and the quantitative limit level of 0.002-0.017 mg/kg were confirmed. In the recovery test in which the standard was treated at a concentration of 0.02 mg/kg, it was confirmed that the proportion of pesticides satisfying the recovery of 70-120% was 85.7-96.4% for each herbal medicine, so it was confirmed that it was a level that could be reviewed by the screening method.

In Vitro Micropropagation of Polygonatum odoratum cv. Gungangbeaksea (둥굴레 '건강백세'의 기내 대량번식)

  • Kim, Man Bae;Park, Chun Geun;Yi, Sookyi;Han, Bong Hee
    • Korean Journal of Plant Resources
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    • v.27 no.5
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    • pp.540-545
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    • 2014
  • The Polygonatum odoratum cv. Gungangbeaksea, bred in Gyeongsangnam-Do Agricutural Research & Extension Service, was cultured in vitro for micropropagate rapidly through the culture of rhizome explants ($5{\times}5mm$). The $7{\times}7mm$ explants of adventitious multi-bud clusters (AMC), obtained through the culture of rhizome explants (MS + 3.0 mg/L BA) were cultured on MS media with BA and TDZ. The shoot multiplication was favorable on the MS medium containing 3.0 mg/L TDZ with 2.8 in shoot number. But the formation of AMC was low in all media tested. The explants of AMC were cultured on MS media containing 1.0~5.0 mg/L TDZ and NAA to multiplicate AMC more. The formation of AMC was a little more stimulated on combined MS media of TDZ and NAA, than that with TDZ alone. The multiplication of shoots and AMC was favorable on MS media with 3.0 mg/L TDZ and 5.0 mg/L NAA, and 5.0 mg/L TDZ and 3.0 mg/L NAA. As the concentration of MS salts increased, the formation of AMC was decreased. But the formation of AMC was more stimulated, as the concentration of sucrose increased to 7%. Therefore, the multiplication of shoots and AMC was suitable on media containing 3.0~5.0 mg/L TDZ and NAA, and 7% of sucrose. The explants of AMC were rooted on media with 3.0 mg/L IBA, or 2.0 mg/L NAA with more than 80% in rooting ratio. The plantlets were treated at $5^{\circ}C$ for 8 weeks, and cultured ex vitro for 8 weeks. The survival ratio of plantlets were 100% in vermiculite, and the mixed soil with perlite 1 volumn and vermiculite 1 volumn.

Shoot Regeneration from the Leaf Explants of Tetragonia tetragonoides $O.\;K_{UNTZE}$ (번행초의 잎 절편으로부터 신초의 재분화)

  • Hwang, Sung-Jin;Pyo, Byoung-Sik;Hwang, Baik
    • Korean Journal of Medicinal Crop Science
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    • v.10 no.2
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    • pp.116-119
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    • 2002
  • A protocol has been developed for differentiation of adventitious shoots directly from leaf segments of Tetragonia tetragonoides O. Kuntze. Murashige and Skoog (MS) medium supplemented with 2 mg/L $N^6-benzyladenine$ (BA) and 0.5 mg/L ${\alpha}-naphthaleneacetic$ acid (NAA) supported the induction of adventitious shoots from leaf explants. Adventitious shoots were multiplied by subculturing on the double strength MS (2MS) medium supplemented with 0.5 mg/L NAA and 2 mg/L BA. Shoots were rooted on MS basal medium without any growth regulators.

Determination and Monitoring of Grayanotoxins in Honey Using LC-MS/MS (LC-MS/MS를 이용한 벌꿀 중 grayanotoxin 분석법 연구 및 실태조사)

  • Lee, Sook-Yeon;Choi, Youn-Ju;Lee, Kang-Bong;Cho, Tae-Yong;Kim, Jin-Sook;Son, Young-Wook;Park, Jae-Seok;Im, Sung-Im;Choi, Hee-Jung;Lee, Dong-Ha
    • Korean Journal of Food Science and Technology
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    • v.40 no.1
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    • pp.8-14
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    • 2008
  • This study was performed to establish analysis methods, and evaluated for grayanotoxin in domestic/foreign honey and wild honey. The molecular weight of grayanotoxins I, II and III, excluding grayanotoxin III that has been commercialized, were analyzed by LC-MS/MS. Then, the molecular structure of grayanotoxins I and II were analyzed by NMR. A total 111 samples (25 Korean honey, 21 Korean wild honey, 13 Korean honeycomb honey, 44 foreign honey, 8 foreign wild honey) were examined to determined whether or not each sample contained grayanotoxins I, II, and III. The honey samples were mixed with methanol and loaded into a tC18 cartridge, the filtrate was diluted with water, and the mixture was then analyzed by ESI triple-quadrupole LC-MS/MS. Grayanotoxins were only found in the foreign wild honey and were not detected in Korean honey, Korean honeycomb honey, or Korean wild honey. Three of the samples contained grayanotoxin I, II, and III, and one sample contained only grayanotoxins I and III. The lowest level for grayanotoxin I was 3.13 ${\pm}$ 0.00 mg/kg, and the highest level was 12.93 ${\pm}$ 0.01 mg/kg. The levels of grayanotoxin II were 0.84 ${\pm}$ 0.01 mg/kg, 0.92 ${\pm}$ 0.00 mg/kg and 1.08 ${\pm}$ 0.01 mg/kg, respectively. The lowest level of grayanotoxin III was 0.25 ${\pm}$ 0.01 mg/kg and the highest level was 3.29 ${\pm}$ 0.74 mg/kg. Through this study, safety management for foreign wild honey has been enabled.

Use of Microsatellite Markers Derived from Genomic and Expressed Sequence Tag (EST) Data to Identify Commercial Watermelon Cultivars (수박 시판 품종의 식별을 위한 Genomic과 Expressed Sequence Tag (EST)에서 유래된 Microsatellite Marker의 이용)

  • Kwon, Yong-Sham;Hong, Jee-Hwa;Kim, Du-Hyun;Kim, Do-Hoon
    • Horticultural Science & Technology
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    • v.33 no.5
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    • pp.737-750
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    • 2015
  • This study was carried out to construct a DNA profile database for 102 watermelon cultivars through the comparison of polymorphism level and genetic relatedness using genomic microsatellite (gMS) and expressed sequence tag (EST)-microsatellite (eMS) markers. Sixteen gMS and 10 eMS primers showed hyper-variability and were able to represent the genetic variation within 102 watermelon cultivars. With gMS markers, an average of 3.63 alleles per marker were detected with a polymorphism information content (PIC) value of 0.479, whereas with eMS markers, the average number of alleles per marker was 2.50 and the PIC value was 0.425, indicating that eMS detects a lower polymorphism level compared to gMS. Cluster analysis and Jaccard's genetic distance coefficients using the unweighted pair group method with arithmetic average (UPGMA) based on the gMS, eMS, and combined data sets showed that 102 commercial watermelon cultivars could be categorized into 6 to 8 major groups corresponding to phenotypic traits. Moreover, this method was sufficient to identify 78 out of 102 cultivars. Correlation analysis with Mantel tests for those clusters using 3 data sets showed high correlation ($r{\geq}0.80$). Therefore, the microsatellite markers used in this study may serve as a useful tool for germplasm evaluation, genetic purity assessment, and fingerprinting of watermelon cultivars.

Multiresidue Analysis of 124 Pesticides in Soils with QuEChERS extraction and LC-MS/MS (QuEChERS 및 LC-MS/MS를 이용한 토양 중 124종 잔류농약다성분 분석법)

  • Gwon, Ji-Hyeong;Kim, Taek-Kyum;Seo, Eun-Kyung;Hong, Su-Myeong;Kwon, Hye-Yong;Kyung, Ki-Sung;Kim, Jang-Eok;Cho, Nam-Jun
    • The Korean Journal of Pesticide Science
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    • v.18 no.4
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    • pp.296-313
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    • 2014
  • A QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) multiresidue method was developed for the simultaneous analysis of 124 pesticides in soil by LC-MS. The procedure involved liquid extraction of soil immersed with 0.2N $NH_4Cl$ by acetonitrile with 1% acetic acid, followed by anhydrous $MgSO_4$ and sodium acetate, and dispersive SPE cleanup with $MgSO_4$, primary secondary amine (PSA) and $C_{18}$. The extracts were analyzed with LC-MS/MS in ESI positive mode. Standard calibration curves were made by matrix matched standards and their correlation coefficients were higher than 0.99. Recovery studies for the validation were carried out using two type soils, loam and sandy loam, at four concentration levels (0.005, 0.01, 0.02, and 0.1 mg/kg). The recoveries of pesticides were in the range of 70-120% with < 20% RSD except 4 pesticides, Benfuracarb, Ethiofencarb, Pymetrozine, and Pyrethrin. This result indicated that the method using QuEChERS and LC-MS/MS could be applied for the simultaneous determination of pesticide residues in soils.