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Electrospray-Mass Spectrometric Analysis of Plasma Pyrophosphates Separated on a Multi-Modal Liquid Chromatographic Column

  • Lee, Su-Hyeon;Lee, Jeong-Ae;Lee, Won-Yong;Chung, Bong-Chul;Choi, Man-Ho
    • Mass Spectrometry Letters
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    • v.2 no.4
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    • pp.92-95
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    • 2011
  • Pyrophosphates are the key intermediates in the biosynthesis of isoprenoids, and their concentrations could reveal the benefits of statins in cardiovascular diseases. Quantitative analysis of five pyrophosphates, including isopentenyl pyrophosphate (IPP), dimethylallyl pyrophosphate (DMAPP), geranyl pyrophosphate (GPP), farnesyl pyrophosphate (FPP), and geranylgeranyl pyrophosphate (GGPP), was performed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) in negative ionization mode. After dilution with methanol, samples were separated on a 3 ${\mu}m$ particle multi-modal $C_{18}$ column ($50{\times}2$ mm) and quantified within 10 min. The gradient elution consists of 10 mM ammonium bicarbonate and 0.5% triethylamine (TEA) in water and 0.1% TEA in 80% acetonitrile was used at the flow rate of 0.4 mL/min. Overall recoveries were 51.4-106.6%, while the limit of quantification was 0.05 ${\mu}g$/mL for GPP and FPP and 0.1 ${\mu}g$/mL for IPP, DMAPP, and GGPP. The precision (% CV) and accuracy (% bias) of the assay were 1.9-12.3% and 89.6-111.8%, respectively, in 0.05-10 ${\mu}g$/mL calibration ranges ($R^2$ > 0.993). The devised LC-MS/MS technique with the multi-modal $C_{18}$ column can be used to estimate the biological activity of pyrophosphates in plasma and may be applicable to cardiovascular events with cholesterol metabolism as well as the drug efficacy of statins.

Quantitative Analysis of Normal Salivary Gland Function using Salivary Scintigraphy (타액선 신티그라피를 이용한 정상 타액선기능의 정량분석)

  • Kim, Hui-Jin;Ko, Myung-Yun;Park, June-Sang
    • Journal of Oral Medicine and Pain
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    • v.25 no.4
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    • pp.345-353
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    • 2000
  • This study was to investigate normal salivary flow rates and normal indices of Quantitative analysis of salivary scintigraphy. 96 adult volunteers were studied by Questionnaire evaluating salivary conditions and clinical examinations. 35(male 23, female 12, age range 23-31years) that absented subjective and objective symptoms related saliva were classified as normal group. The normal group underwent measurement unstimulated and stimulated salivary flow rates and salivary scintigraphy. The obtained results were as follows: 1. There were not significant in sex differences of unstimulated and stimulated salivary flow rates. The unstimulated salivary flow rate was $0.66{\pm}0.41g/min$, stimulated salivary flow rates was $1.61{\pm}0.69g/min$. 2. As comparing of parameters of salivary scintigraphy, the Uptake ratio(UR), $T_{max}$, $T_{min}$, Maximum accumulation (MA), Maximum secretion(MS) of parotid and submandibular glands were not significant in sex and side-ralated differences. 3. The UR, $T_{max}$, MA, MS of parotid gland were significantly higher than those of submandibular gland; in the parotid gland, UR, $3.67{\pm}0.88$, $T_{max}$, $18.77{\pm}0.43min$, MA, $41.35{\pm}9.22%$, MS, $43.13{\pm}9.13%$; in the submandibular gland, UR, $3.04{\pm}0.10$, $T_{max}$, $18.48{\pm}0.52min$, MA, $36.47{\pm}14.18%$, MS, $36.88{\pm}12.20%$. 4. As classifying of time-activity curve, the most of parotid gland was N-type(97.1%), submandibular gland was observed in order of M-type(67.1%), N-type(21.4%), F-type(11.4%), however, was not observed S-type. 5. As the type of time-activity curve of submandibular gland was more flattened, the UR, $T_{max}$, MA, MS were significantly decresed.

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Interaction Between time of Nodal Explant Collection and Growth Regulators Determines the Efficiency of Morus alba Micropropagation

  • Hassanein A.M.;Galal A.A.;Azooz M.M.
    • Journal of Plant Biotechnology
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    • v.5 no.4
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    • pp.225-231
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    • 2003
  • The hormonal requirement suiting micropropagation of Morus alba during any season throughout the year was studied. Sprouting frequency from axillary buds of M. alba was greatly influenced by the time of explant collection, the highest value was achieved when nodal explants were collected at the end of bud dormancy period (late in March) and cultured on Murashige and Skoog (MS) medium supplemented with low concentration (0.5 mg/L) of BAP, kinetin or IBA (85-68%). In addition, they showed higher axillary bud sprouting on growth-regulators-free medium (49%) than others collected in autumn or winter and cultured on medium supplemented with various growth regulators (47-48%). Regardless of that period, young explants with greenish buds collected in summer exhibiting high sprouting frequency (66%) on MS medium supplemented with 0.5 mg/L kinetin and 0.5 mg/L GA3. Shoot multiplication via adventitious bud formation was achieved when the nodal explants were cultured on MS medium supplemented with 2 mg/L BAP and 0.2 mg/L IBA. Further multiplication via nodal explants of in vitro grown shoots was obtained on MS medium supplemented with 0.5 mglL BAP and 0.5 mg/L GA3. While half strength MS medium supplemented with low concentration (0.5 mg/L) of IBA, IAA or 2,4-D stimulated adventitious root formation, IBA was the best. After transfer the plantlets to the soil, acclimatization for three weeks was essential prerequisite for survival in high frequency (92%). Peroxidase activity is related to break of bud dormancy where maximum enzyme activity was detected when the lateral buds were induced to commence growth under field condition (early in spring) or in vitro.

Callus induction and high-efficiency plant regeneration via somatic embryogenesis in Papaver nudicaule L., an ornamental medicinal plant

  • Yang, Jing Li;Zhao, Bo;Seong, Eun-Soo;Kim, Myong-Jo;Kang, Won-Hee;Kim, Na-Young;Yu, Chang-Yeon;Li, Cheng Hao
    • Plant Biotechnology Reports
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    • v.4 no.4
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    • pp.261-267
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    • 2010
  • We describe culture conditions for a high-efficiency in vitro regeneration system of Papaver nudicaule through somatic embryogenesis and secondary somatic embryogenesis. The embryogenic callus induction rate was highest when petiole explants were cultured on Murashige and Skoog (MS) medium containing 1.0 mg $1^{-1}$ ${\alpha}$-naphthaleneacetic acid (NAA) and 0.1 mg $1^{-1}$ 6-benzyladenine (BA) (36.7%). When transferred to plant growth regulator (PGR)-free medium, 430 somatic embryos formed asynchronously from 90 mg of embryogenic callus in each 100-ml flask. Early-stage somatic embryos were transferred to MS medium containing 1.0 mg $1^{-1}$ BA and 1.0 mg $1^{-1}$ NAA to germinate at high frequency (97.6%). One-third-strength MS medium with 1.0% sucrose and 1.0 mg $1^{-1}$ $GA_3$ had the highest frequency of plantlet conversion from somatic embryos (91.2%). Over 90% of regenerated plantlets were successfully acclimated in the greenhouse. Secondary somatic embryos were frequently induced directly when the excised hypocotyls of the primary somatic embryos were cultured on MS medium without PGRs. Sucrose concentration significantly affected the induction of secondary embryos. The highest induction rate (89.5) and number of secondary somatic embryos per explant (9.3) were obtained by 1% sucrose. Most secondary embryos (87.2-94.3%) developed into the cotyledonary stage on induction medium. All cotyledonary secondary embryos were converted into plantlets both in liquid and on semisolid 1/3-strength MS medium with 1.0% sucrose.

Improved Preimplantation Development of Cloned Porcine Embryos through Supplementation of Histone Deacetylase Inhibitor MS-275

  • Fang, Xun;Qamar, Ahmad Yar;Shin, Sang Tae;Cho, Jongki
    • Journal of Veterinary Clinics
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    • v.36 no.5
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    • pp.253-258
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    • 2019
  • The objective of this study was to analyse the effects of MS-275 (Class I and II histone deacetylase inhibitor) supplementation on the development of porcine in-vitro somatic nuclear transfer embryo production. During in-vitro development, early embryos were exposed to different concentrations of MS-275 (0, $5{\mu}M$, $10{\mu}M$, and $20{\mu}M$). In in-vitro culture supplemented group, the blastocyst development rate was significantly enhanced by $10{\mu}M$ concentration than other groups (24.0% vs. 19.3%, 21.8%, 11.5%; P < 0.05). Additionally, the 6 h supplementation group, significantly improved the blastocysts production than 24 h, 48 h and control groups (26.1% vs. 17.0%, 15.2%, 2.8%; P < 0.05). Following supplementation with optimal concentrations and time ($10{\mu}M$-6 h group), the blastocyst production was significantly higher than control (25.7% vs 15.8%; P < 0.05). The optimal concentrations of MS-275 significantly enhanced the percentages of ICM:TE than control (43.6% vs. 38.4%; P < 0.05) accompanied with significantly higher expression levels of reprogramming related genes (POU5F1, Naong, and SOX2). In conclusion, the optimal concentrations of $10{\mu}M$ MS-275 and 6 h supplementation during in-vitro culture can significantly improve the quality of porcine in-vitro somatic nuclear transfer embryos through histone acetylation and epigenetic modification. Increasing the efficiency of clonal animal production will greatly promote the development of animal disease models and xenotransplantation.

Analytical Methods of Fenpyroximate in Herbal Medicines (생약 중 Fenpyroximate의 분석법 연구)

  • Lee, Ju-Hee;Lee, Yoon-Jeong;Kang, In-Ho;Kim, Do-Hoon;Kang, Shin-Jeong;An, Kyung-A;Lee, Ryun-Kyung;Suh, Sang-Chul;Lee, Jeong-Rim
    • The Korean Journal of Pesticide Science
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    • v.18 no.3
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    • pp.141-147
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    • 2014
  • Fenpyroximate is acaricide of pyrazole group. This acaricide have already been permitted for herb cultivation. This experiment was conducted to establish a determination method for fenpyroximate residue in herbal medicines using HPLC-PDA and HPLC-MS/MS. Fenpyroximate residue was extracted with acetone from samples of herbal which Liquorice Root (Glycyrrhiza uralensis) and Safflower Seed (Carthamus tinctorius Linne). The extract was diluted with saturated saline water and dichloromethane liquid-liquid partition (extraction) was followed to recover fenpyroximate from the aqueous phase. Amino propyl ($NH_2$) and florisil column chromatography was additionally employed for final clean up of the extract. The fenpyroximate was quantitated by HPLC-PDA and HPLC-MS/MS. The herbals were fortified with fenpyroximate at 2 or 3 levels per crop. Mean recovery ratio were ranged from 72.0 to 106.4%. The coefficients of variation were ranged from 0.2 to 4.4. Therefore, this analytical method was reproducible and sensitive enough to determine the residue of fenpyroximate in herbal medicines.

Analysis of Nitrosamines Concentration in Condom by using LC-MS/MS (LC-MS/MS를 이용한 콘돔에 함유된 니트로사민류 농도 분석)

  • Park, Na-Youn;Kim, Sungmin;Jung, Woong;Kho, Younglim
    • Journal of the Korean Chemical Society
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    • v.62 no.3
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    • pp.181-186
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    • 2018
  • Nitrosamines are the nitrosocompounds which are produced by nitrosation reactions of the secondary amine and nitrite, and has been found to be produced through the vulcanization process during the production of rubber products Recently, nitrosamines have been detected in rubber products and become a major topic. Condoms are disposable medical devices, so safety is important because they come into direct contact with the skin and mucous membranes. In this study, we developed an analytical method for nitrosamines in condoms by applying ISO 29941 method. The samples were eluted by distilled water, and target compounds were extracted by liquid-liquid extraction with dichloromethane. And then after concentrated, and quantitatively analyzed by LC-MS/MS. The accuracies of the analytical method were ranged from 85.8 to 108.7%, precisions were lower than 11.5%, and the detection limits were from 0.11 (NDPA and NDBA) to 0.48 (NPYR) ng/mL. Among the 31 condom samples, NDBA was detected from 2 cases by extraction of distilled water, and NDMA were detected from 1 case, NDEA from 4 cases and NDBA from 26 cases by extraction of artificial saliva (pH 4.5). The total amount of nitrosamines in all samples were less than $500{\mu}g/kg$.

A Fast Determination of Globotriaosylsphingosine in Plasma for Screening Fabry Disease Using UPLC-ESI-MS/MS

  • Yoon, Hye-Ran
    • Mass Spectrometry Letters
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    • v.6 no.4
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    • pp.116-119
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    • 2015
  • Globotriaosylsphingosine (lyso-Gb3) is considered as one of the biological marker for Fabry disease. To date, a reliable biomarker that reflects disease severity and progression has not been discovered to guide the management of Fabry disease. A new method included a simple protein precipitation with acetonitrile in 100 μL of plasma following analyte separation on an Phenomenex Kintex- C18 column using a gradient elution (0.1% formic acid in 5-90% acetonitrile). Total run time was within 12 min including sample preparation and MS/MS analysis. The limit of detection and limit of quantitation were 1 ng/mL and 2 ng/mL, respectively. The calibration curve was linear over the concentration range of 2.0-200.0 ng/mL (r2 = 0.9999). Inter-day accuracy and precision at 7 level were 93.4-100.7% with RSD of 0.55-5.97%. Absolute recovery was 97.6-98.6%. The method was applied to human and mice plasma, proved the suitability for quantification of lyso-Gb3 for screening, diagnosis and therapeutic monitoring of Fabry disease patients.

Determination of agrochemical residues in tobacco using matrix solid-phase dispersion and GC/MS

  • Lee, Jeong-Min;Min, Hye-Jeong;Park, Jin-Won;Lee, Moon-Young;Jang, Gi-Chul
    • Journal of the Korean Society of Tobacco Science
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    • v.36 no.1
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    • pp.12-19
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    • 2014
  • A matrix solid-phase dispersion (MSPD) method was developed for extracting and cleaning-up the selected agrochemicals in tobacco using gas chromatography-mass spectrometry with selected ion monitoring (GC/MS-SIM). Different parameters of the method were investigated and optimized, such as the type of solid-phase (alumina, $C_{18}$ and Florisil) and eluent (acetone, acetonitrile, ethylacetate and n-hexane). The best results were obtained using 0.5 g of tobacco sample, 1.0 g of $C_{18}$ as dispersant sorbent, 1.0 g of Florisil as clean-up sorbent and acetonitile saturated with n-hexane as eluting solvent. The method was validated using tobacco samples fortified with agrochemicals at their different concentration levels. This method gave good linearity for the selected agrochemicals of ranging from $0.01{\mu}g/mL$ to $0.1{\mu}g/mL$. Recoveries of the selected agrochemicals in tobacco were more than 80 % and reproducibilities were found to be better than 10 % RSD. Those results suggested that the analytical procedure including MSPD method in combining with GC/MS could be applicable to the rapid determination often the selected agrochemicals in tobacco.

Rapid Quantification of Topotecan in Biological Samples by Liquid Chromatography/Tandem Mass Spectrometry

  • Shin, Beom-Soo;Lee, Mann-Hyung;Yoo, Sun-Dong
    • Journal of Pharmaceutical Investigation
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    • v.39 no.5
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    • pp.367-372
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    • 2009
  • A rapid liquid chromatography/tandem mass spectrometry (LC/MS/MS) assay method was developed for the determination of topotecan levels in rat serum. The assay utilized a single liquid-liquid extraction with a mixture of ethy l acetate and acetonitrile (6:1 v/v) and isocratic elution. The multiple reaction monitoring was based on the transition of m/z 422.0$\rightarrow$376.5 for topotecan and 315.1$\rightarrow$226.6 for clomipramine (internal standard). The developed assay was validated to demonstrate the specificity, recovery, lower limit of quantification (LLOQ), accuracy and precision. The assay was linear over a concentration range from 0.5-100 ng/mL, with LLOQ being 0.5 ng/mL using a small volume of rat serum (0.1 mL). The mean intra- and inter-day assay accuracy was 87.7-111.0% and 97.8-108.3, respectively, and the mean intra- and interday precision was between 1.6-4.3% and 3.8-10.3, respectively. The developed assay was applied to a pharmacokinetic study after a bolus i.v. injection of topotecan in rats.