Simultaneous analysis of mono-, di-, and tri-ethanolamine in cosmetic products using liquid chromatography coupled tandem mass spectrometry

신경오,이용문 대한약학회 2016 Archives of Pharmacal Research Vol.39 No.1

Alkanolamines such as monoethanolamine (MEA), diethanolamine (DEA), and triethanolamine (TEA) are used as wetting agents in shampoos, lotions, creams, and other cosmetics. DEA is widely used to provide lather in shampoos and maintain a favorable consistency in lotions and creams. Although DEA is not harmful, it may react with other ingredients in the cosmetic formula after extended storage periods to form an extremely potent carcinogen called nitrosodiethanolamine (NDEA), which is readily absorbed through the skin and has been linked to the development of stomach, esophagus, liver, and bladder cancers. The purpose of this study was to develop a simultaneous quantification method for measurement of MEA, DEA, and TEA in cosmetic products. Liquid chromatography coupled tandem mass spectrometry (LC–MS/MS) was performed using a hydrophilic interaction liquid chromatography (HILIC) column with isocratic elution containing acetonitrile and 5 mM ammonium formate in water (88:12, v/v). Identification and quantification of alkanolamines were performed using MS/MS monitoring to assess the transition from precursor to product ion of MEA (m/z, 61.1 ? 44.0), DEA (m/z, 106.1 ? 88.0), TEA (m/z, 150.1 ? 130.0), and the internal standard triethylamine (m/ z, 102.2 ? 58.0). Alkanolamines extractions were simplified using a single extraction with acetonitrile in the cosmetic matrix. Performance of the method was evaluated with quality parameters such as specificity, carry-over, linearity and calibration, correlation of determination (R2), detection limit, precision, accuracy, and recovery. Calibration curves of MEA (2.9–1000 ppb), DEA (1–1000 ppb), and TEA (1–1000 ppb) were constructed by plotting concentration versus peak-area ratio (analyte/internal standard with a correlation coefficient greater than 0.99). The intra- and inter-assay accuracy ranged from 92.92 to 101.15 % for all analytes. The intra- and inter-assay precision for MEA, DEA, and TEA showed all coefficients of variance were less than 9.38 % for QC samples. Limits of detection and limits of quantification were 2.00 and 15.63 ppb forMEA, 0.49 and 1.96 ppb for DEA, and 0.49 and 1.96 ppb for TEA, respectively. This novel quantification method simplified sample preparation and allowed accurate and reproducible quantification of alkanolamines in the ng/g cosmetic weight (ppb) range for several cosmetic products.

Ginsenoside Rb1 Enhances Keratinocyte Migration by a Sphingosine-1-Phosphate-Dependent Mechanism

신경오,최성재,Yoshikazu Uchida,김인용,정윤화,박경호 한국식품영양과학회 2018 Journal of medicinal food Vol.21 No.11

The cutaneous wound healing process is tightly regulated by a range of cellular responses, including migration. Sphingosine-1-phosphate (S1P) is a signaling lipid produced in keratinocytes (KC) and it is known to stimulate skin wound repair through increased KC migration. Of the multifunctional triterpene ginsenosides, Rb1 enhances cutaneous wound healing process by increasing KC migration, but cellular mechanisms responsible for the Rb1-mediated increase in KC migration are largely unknown. Therefore, we hypothesized that, and assessed whether, Rb1 could stimulate KC migration through S1P-dependent mechanisms. Rb1 significantly increases S1P production by regulating the activity of metabolic conversion enzymes associated with S1P generation and degradation, sphingosine kinase 1 (SPHK1) and S1P lyase, respectively, in parallel with enhanced KC migration. However, blockade of ceramide to S1P metabolic conversion using a specific inhibitor of SPHK1 attenuated the expected Rb1-mediated increase in KC migration. Furthermore, a pan-S1P receptor inhibitor pertussis toxin significantly attenuated Rb1-induced stimulation of KC migration. Moreover, the Rb1-induced increases in KC migration required S1P receptor(s)-mediated activation of ERK1/2 and NF-κB, leading to production of key cutaneous migrating proteins, matrix metalloproteinase (MMP)-2 and MMP-9. Taken together, the results show that Rb1 stimulates KC migration through an S1P→S1P receptor(s)→ERK1/2→NF-κB→MMP-2/-9 pathway. This research revealed a previously unidentified cellular mechanism for Rb1 in enhancing KC migration and pointing to a new therapeutic approach to stimulate the cutaneous wound healing process.

Ginsenoside compound K inhibits angiogenesis via regulation of sphingosine kinase-1 in human umbilical vein endothelial cells

신경오,서초희,조효현,오세관,홍선표,유환수,홍진태,오기완,이용문 대한약학회 2014 Archives of Pharmacal Research Vol.37 No.9

Ginsenoside compoundK(CK) is a metabolite ofthe protopanaxadiol-type saponins of Panax ginseng C.A. Meyer (Araliaceae), has long been used to treat against thedevelopment of cancer, inflammation, allergies, and diabetes. This study examined the anti-angiogenic properties of CKagainst sphingosine 1-phosphate (S1P)-induced cell migrationvia regulation of sphingosine kinase 1 (SPHK1) inhuman umbilical vein endothelial cells (HUVEC). Studies onS1P-induced cell migration, expression ofSPHK1 andMMPsand analysis of sphingolipidmetabolites byLC–MS/MSwereexamined after the treatment of CK (2.5, 5, 10 lg/mL) inHUVEC. S1P produced by SPHK1 is also involved in cellgrowth, migration, and protection of apoptosis; therefore, wesought to investigate whether ginsenosides are able to regulateSPHK1. For this purpose, we developed an inhibitoryassay of SPHK1 activity and an analytical method fordetection of S1P and other sphingolipid metabolites in HUVEC. Ginsenoside CK inhibited 100 nM S1P-induced cellmigrations in a dose-dependent manner. Among tested ginsenosides,CK exclusively inhibited S1P production, SPHK1activity and SPHK1 expression in HUVEC, whereasexpression of the pro-apoptotic sphingolipids, sphingosineand ceramide, was increased in response to CK. The major subspecies of the increased ceramide was C24:0-ceramide. CKalso disrupted the sphingolipid rheostat, which ultimatelyinfluences cell fate, and dose-dependently inhibited HUVECmigration by reducing expression of metalloproteinases(MMPs). GinsenosideCKacts as a uniqueHUVECmigrationinhibitor by regulating MMP expression, as well as theactivity of SPHK1 and its related sphingolipid metabolites.

Derivatization reagents for Mass Spectrometry

신경오, 이부민, 이용문 충북대학교 약품자원개발연구소 2014 약학논문집 Vol.29 No.-

Liquid chromatography coupled with electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) has been frequently utilized for the sensitive and selective determination of the trace level compounds in biological samples. In LC/ESI-MS/MS, chemical derivatization is sometimes used to enhance the detection sensitivity of the analytes. The electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) sources of MS have contributed to the advancement of LC-MS and LC-MS/MS techniques for the analysis of biological samples. However, one major obstacle is the weak ionization of some analytes in the ESI and APCI techniques. In this review, we introduce high-sensitivity methods using several derivatization reagents for ionization enhancement. We also present an overview of chemical derivatization methods that have been applied to small molecules, such as amino acids and steroids, in biological samples.

Sphingolipid Metabolites Profiling in Hair Growth

신경오 ( Kyong-oh Shin ) 한국피부장벽학회 2018 한국피부장벽학회지 Vol.20 No.2

Sphingolipids, a major component of hair lipids can determine the physicochemical properties of human hairs such as the chemical diffusion barrier, water retention and cell cohesion. In this study, we have developed a quantitation method for sphingolipids in animal and human hair. The lipid contents of sphingoid bases, sphingoid bases 1-phosphate, Ceramide (Cer) and dihydroceramide (DHCer) in hair were measured by liquid chromatography linked tandem mass spectrometry (LC-MS/MS). The established method was evaluated against quality parameters such as specificity, carryover, linearity and calibration, correlation of determination (R2), LOD, LOQ, precision, accuracy, and recovery. Specifically, analyzed DHCer levels were higher in normal hair roots to distal tip ends than Cer contents ranged from 1960.23 to 1373.24 pmol/g. Interestingly, DHCer were decreased significantly in alopecia patient upper hair fiber compared to alopecia patient side hair fiber. Specific ceramides (C16:0 and C24:1) in alopecia patient upper hair were observed in very low levels, while Cer was not change in alopecia hair. In experimental mouse model of alopecia hair and skin, Cer and DHCer were significantly low as well. These results suggested that hair DHCer content may be used as a potential biomarker for the progress in alopecia pathogenesis.

레이저 변위 신호에 의한 숫돌마멸 검출

신경오(K. O. Shin),왕덕현(D. H. Wang),김원일(W. I. Kim),이윤경(Y. K. Lee) 한국정밀공학회 1998 한국정밀공학회 학술발표대회 논문집 Vol.1998 No.5월

흑연/에폭시 복합재료의 엔드밀가공시 발생하는 음향방출신호 특성

정전영,신경오,김원일 慶南大學校 附設 工業技術硏究所 1998 硏究論文集 Vol.15 No.2

본 연구에서눈 흑연/에폭시 복합재료의 엔드밀 가공시 절삭조건의 변화에 따른 공구 마멸현상을 음향방출신호에 의하여 검출하고 엔드밀 마멸이 표면거칠기에 미치는 영향을 연구하였다. 실효치 전압은 절삭속도 증가에 따라 증가하고 같은 절삭조건에서는 이송속도가 낮을 때가 공구 마멸 및 표면거칠기 상태가 양호해짐을 확인하였다. 공구의 플랭크 마멸량, ?? =0.2㎜부근에서 실효치 전압의 상승과 하강이 불규칙적으로 심해지며, 이 때를 적절한 공구교한 시기로 판단할 수 있다. In this study, AE signals used to detect the wear of end-mill on various cutting conditions and investigated the effect of the end-mill wear to the surface roughness. The ?? values increased as the cutting speed increases, and for a given cutting conditions, the decreasing of the federate give a favorable effect to the tool wear and the surface roughness. The ?? values rise and go down rapidly when the flank wear comes to ??= 0.2㎜, thus it can be considered that it is the appropriate time to change the tool.

Vitamin C Stimulates Epidermal Ceramide Production by Regulating Its Metabolic Enzymes

김건표,신경오,박경호,윤혜정,Shivtaj Mann,이용문,조윤희 한국응용약물학회 2015 Biomolecules & Therapeutics(구 응용약물학회지) Vol.23 No.6

Ceramide is the most abundant lipid in the epidermis and plays a critical role in maintaining epidermal barrier function. Overall ceramide content in keratinocyte increases in parallel with differentiation, which is initiated by supplementation of calcium and/or vitamin C. However, the role of metabolic enzymes responsible for ceramide generation in response to vitamin C is still unclear. Here, we investigated whether vitamin C alters epidermal ceramide content by regulating the expression and/or activity of its metabolic enzymes. When human keratinocytes were grown in 1.2 mM calcium with vitamin C (50 mg/ml) for 11 days, bulk ceramide content significantly increased in conjunction with terminal differentiation of keratinocytes as compared to vehicle controls (1.2 mM calcium alone). Synthesis of the ceramide fractions was enhanced by increased de novo ceramide synthesis pathway via serine palmitoyltransferase and ceramide synthase activations. Moreover, sphingosine-1-phosphate (S1P) hydrolysis pathway by action of S1P phosphatase was also stimulated by vitamin C supplementation, contributing, in part, to enhanced ceramide production. However, activity of sphingomyelinase, a hydrolase enzyme that converts sphingomyelin to ceramide, remained unaltered. Taken together, we demonstrate that vitamin C stimulates ceramide production in keratinocytes by modulating ceramide metabolicrelated enzymes, and as a result, could improve overall epidermal barrier function.

Quantification of 4-methylimidazole in carbonated beverages by ultra-performance liquid chromatography-tandem mass spectrometry

조효현,이용문,신경오,서초희,이신희,유환수,윤혜란,김정우 대한약학회 2015 Archives of Pharmacal Research Vol.38 No.7

2- and 4-methylimidazoles (2-MI and 4-MI) are undesired byproducts produced during the manufacture of caramel color used to darken food products such as carbonated beverages. The Office of Environmental Health Hazard Assessment in California listed 4-MI as carcinogen in January 2011 with a proposed no significant risk level at 29 lg per person per day. Thus, a quantitative analytical measurement for 2-MI and 4-MI is desired for reliable risk assessments for exposure. An ultra-performance liquid chromatography (UPLC) coupled tandem mass spectrometric (MS/MS) method was developed for the quantification of 4-MI in beverage samples. Chromatographic separation of 2-MI and 4-MI were achieved by using a PFP reversed-phase column and a stepwise gradient of methanol and distilled water containing 0.1 % formic acid. Identification and quantification of 2-MI and 4-MI were performed using electrospray ionization-tandem mass monitoring the precursor to product ion transitions for 2-MI at m/z 83.1 ? 42.2 and 4-MI at m/z 83.1 ? 56.1 with melamine at m/z 127.1 ? 85.1 as the internal standard. The performance of the method was evaluated against validation parameters such as specificity, carryover, linearity and calibration, correlation of determination (r2), detection limit, precision, accuracy, and recovery. Calibration curves at 10–400 ng/mL were constructed by plotting concentration versus peak-area ratio (analyte/ internal standard) and fitting the data with a weighted 1/x. The accuracy of the assay ranged from 93.58 to 110.53 % for all analytes. Intra-assay precision for 2-MI and 4-MI were below 7.28 (relative standard deviation/RSD %) at QC samples. Here we present a new and improved method using UPLC-MS/MS to significantly simplify sample preparation and decrease chromatographic run time. This method allows accurate and reproducible quantification of 4-MI in carbonated beverages as low as sub ng/mL (ppb) levels.

Echinacea purpurea root extract enhances the adipocyte differentiation of 3T3-L1 cells

신동미,최경미,이윤선,김원균,신경오,오세관,정재철,이미경,이용문,홍진태,윤여표,유환수 대한약학회 2014 Archives of Pharmacal Research Vol.37 No.6

Echinacea purpurea has been shown to haveanti-diabetic activities; for example, it activates peroxisomeproliferator-activated receptor c (PPARc) and increasesinsulin-stimulated glucose uptake. Adipogenesis has beenused to study the insulin signaling pathway and to screenanti-diabetic compounds. The present study was conductedto investigate the effects of an ethanol extract of E. purpurea(EEEP) and its constituents on the insulin-induced adipocytedifferentiation of 3T3-L1 preadipocytes. When adipocytedifferentiation was induced with insulin plus 3-isobutyl-1-methylxanthine and dexamethasone, the accumulation oflipid droplets and the cellular triglyceride content weresignificantly increased by EEEP. The expressions of PPARcand C/EBPa in adipocytes treated with EEEP were graduallyincreased as compared with control cells. Fat accumulationand triglyceride content of adipocytes treated with dodeca-2(E),4(E)-dienoic acid isobutylamide were significantlyincreased as compared with control cells. Theexpressions of PPARc and C/EBPa in adipocytes treatedwith dodeca-2(E),4(E)-dienoic acid isobutylamide weresignificantly higher than in control cells. These results suggestEEEP promotes the adipogenesis that is partiallyinduced by insulin and that dodeca-2(E),4(E)-dienoic acidisobutylamide appears to be responsible for EEEP-enhancedadipocyte differentiation.