HPLC-MS/MS를 이용한 트리클로로에틸렌 대사산물의 다중 분석법 확립

안영아,고영림,이승호,신미연,전중대,김성균,Ahn, Youngah,Kho, Younglim,Lee, Seungho,Shin, Mi-Yeon,Jeon, Jung Dae,Kim, Sungkyoon 한국환경보건학회 2014 한국환경보건학회지 Vol.40 No.2

Objectives: We aimed to develop a measurement method of five metabolites of trichloroethylene (TCE) in a concurrent biological sample, e.g., trichloroacetic acid (TCA), dichloroacetic acid (DCA), S-(1,2-dichlorovinyl) glutathione (DCVG), S-(1,2-dichlorovinyl)-L-cysteine (DCVC), and N-Acetyl-S-(1,2-dichlorovinyl)-L-cysteine (NAcDCVC) and to validate the method before application to pharmacokinetic study. Methods: TCE metabolites were simultaneously analyzed using high performance liquid chromatography coupled with electrospray ionization mass spectrometry (HPLC-ESI-MS/MS) with as little as 50 ${\mu}L$ of serum and urine. DCA, TCA and NAcDCVC were extracted with diethyl ether, while DCVC and DCVG were extracted by solid phase extraction. This method was validated according to the guidelines for bioanalytical method validation of the Korean National Institute of Toxicological Research. Then, we determined the five metabolites in five strains of mice at 24 hr after exposure to 1 g TCE /kg body weight. Results: The limits of detection for the five metabolites in biological samples ranged from 0.001 to 0.076 nmol/mL, which is comparable to or better than those previously reported. Most calibration curves showed good linearity ($R^2=0.99$), and between-batch variation was less than 20% expressing acceptable robustness and reproducibility. Using this method, we found TCA and DCA were detected in all test mice at 24 hr after the oral administration while NAcDCVC and DCVC were detected in some strains, which showed strain-dependent metabolism of TCE. Conclusions: The present method could provide robust and accurate measurements of major key metabolites of TCE in biological media, which allowed concurrent analysis of TCE metabolism for limited amounts of biospecimens.

LC-MS/MS 시스템을 이용한 소변 중 N-니트로사민류 분석법 확립

박나연,정웅,고영림,Park, Na-Youn,Jung, Woong,Kho, Younglim 대한화학회 2017 대한화학회지 Vol.61 No.2

N-니트로사민은 이차 아민과 아질산이 산성조건 하에서 니트로소화 반응을 통해 생성되는 니트로소 화합물이다. 약 300여종이 존재하며, 그 중 90%가 동물실험을 통해 발암성이 있음이 확인되었다. 1987년 IARC에서 NDMA와 NDEA를 Group 2A로 지정하였고, NDPA, NDBA, NPYR, NPIP, NMOR을 Group 2B로 지정하였다. 본 연구에서는 N-니트로사민류의 생물학적 모니터링을 위하여 소변 중 N-니트로사민류의 분석법을 확립하였다. 소변시료는 고체상추출(Solid phase extraction, SPE)을 통하여 전처리 한 후, LC-(APCI)-MS/MS를 이용하여 정량분석 하였다. 확립된 분석법의 정확도는 85.8~110.2% 이었고, 정밀도는 1.1~10.5%로 나타났다. 검출한계는 0.0002 (NDBA) ~ 0.0793 (NDMA) ng/ml 이었고, 검량선 회귀식의 상관계수($r^2$)은 0.999 이상으로 우수한 직선성을 보여주었다. 실제 소변 중 N-니트로사민류의 평균 농도는 NDMA 2.645 mg/g creatinine, NDEA 0.067 mg/g creatinine, NMEA 0.009 mg/g creatinine, NDBA 0.011 mg/g creatinine, NPIP 0.271 mg/g creatinine, NPYR 0.413 mg/g creatinine 이고, NDPA와 NMOR은 검출되지 않았다. 추후 N-니트로사민류의 인체 노출량 평가 및 위해평가를 위한 기기분석방법으로 활용될 수 있을 것으로 판단된다. N-nitrosamines are the nitroso compounds which are produced by nitrosation reactions of the secondary amine and nitrite under acidic conditions. Approximately 300 species of N-nitrosamine have been tested for carcinogenicity in laboratory experiments, with 90% of them demonstrated carcinogenic effects different animal species, including higher primates. In 1978, IARC classified NDMA and NDEA as Group 2A, and NDPA, NDBA, NPIP, NPYR and NMOR as Group 2B. In this study, we established pretreatment and analytical method for N-nitrosamines (NDMA, NDEA, NMEA, NDPA, NDBA, NPIP, NPYR and NMOR) in human urine for biological monitoring of N-nitrosamines. The analytes were extracted using solid phase extraction (SPE), then quantitative analysis was performed by LC-(APCI)-MS/MS. The accuracies of the established method were between 85.8~108.7% and precisions were lower than 20%. The limit of detection (LOD) were between 0.0002 (NDBA) and 0.0793 (NDMA) ng/ml. The linearity obtained was satisfying for the 8 N-nitrosamines, with a coefficient of determination ($r^2$) higher than 0.999. The mean concentrations of N-nitrosamines in the urine were 2.645 mg/g creatinine for NDMA, 0.067 mg/g creatinine for NDEA, 0.009 mg/g creatinine for NMEA, 0.011 mg/g creatinine for NDBA, 0.271 mg/g creatinine for NPIP and 0.413 mg/g creatinine for NPYR. NDPA and NMOR were not detected. It can be used as a instrumental methodology for evaluation and risk assessment of human exposure to N-nitrosamines for the further research.

LC-MS/MS 시스템을 이용한 모유 중 파라벤류 분석법 확립

박나연,이은희,고영림,Park, Na-Youn,Lee, Eun-Hee,Kho, Younglim 대한화학회 2016 대한화학회지 Vol.60 No.2

Parabens were commonly used for preventing the growth of microorganisms as preservatives in the pharmaceutical, cosmetic and food industry. Also, parabens are known endocrine disruptors because of their estrogenic effects on human. Parabens affect the endocrine system and show adverse effect such as, genital malformations, precocious puberty and testicular cancer in young children, infants and fetuses. In this study, we developed analytical method for four parabens (methyl paraben, ethyl paraben, propyl paraben, butyl paraben) in human breast milk which frequently consumed by newborn baby. The analytes were extracted using liquid-liquid extraction (LLE) after enzyme hydrolysis with protease and lipase, then quantitative analysis was performed by liquid chromatography tandem mass spectrometry (LC-MS/MS). The method validation results were as follows; the linearity of calibration curves were excellent with coefficient of determinations (r²) higher than 0.999, the limit of detections (LODs) were 0.019~0.044 ng/mL, the accuracies were 85.3~105.9% and the precisions were lower than 10%. The average concentration ± standard deviation of parabens in ten human breast milk sample were MP 0.660 ± 0.519 ng/mL, EP 1.631 ± 2.081 ng/mL and PP 0.326 ± 0.320 ng/mL, and BP was not detected. 파라벤은 화장품, 식품, 의약품 등에 첨가되는 살균성 보존제로 미생물의 성장을 억제한다. 그러나 체내에서 에스트로겐과 유사한 작용을 하는 내분비계 교란물질이며, 특히 독성물질에 민감한 영유아 및 태아에게 노출될 경우 생식기 기형, 고환암, 성조숙증 등 성장발달에 큰 영향을 미친다. 따라서 본 연구에서는 영아가 주로 섭취하는 모유에서의 파라벤 수준을 확인하기 위하여 모유 중 4가지 파라벤(methyl paraben, ethyl paraben, propyl parabne, butyl paraben)의 분석법을 확립하였다. 시료는 액체상추출(Liquid-liquid extraction, LLE)을 통하여 전처리 한 후, 액체크로마토그래피 텐덤 질량분석기(liquid chromatography tandem mass spectrometry, LC-MS/MS)를 사용하여 정량분석 하였다. 확립된 분석조건에서 얻어진 검량선의 직선성은 r² > 0.999이고, 검출한계(LOD)는 0.019~0.044 ng/mL이었다. 또한 정확도는 85.3~105.9%, 정밀도는 10% 이하의 값을 나타내었다. 10개의 모유시료 중 파라벤류의 평균농도 ± 표준편차는 MP 0.660 ± 0.519 ng/mL, EP 1.631 ± 2.081 ng/mL, PP 0.326 ± 0.320 ng/mL이었고, BP는 불검출 되었다.

국내의 조리 식품 중 과불화화합물의 잔류 수준

신재호(Jae-Ho Shin),고영림(Younglim Kho),박나연(Nayoun Park),방선아(Sunah Bang),임초롱(Cho Rong Lim),김판기(Pan-Gyi Kim),김정환(Jung Hoan Kim) 한국식품과학회 2016 한국식품과학회지 Vol.48 No.6

국내에서 유통되는 조리식품 6종류, 151점의 시료를 수집하여 LC-MS/MS를 이용해 PFCs 잔류 수준을 측정하였다. 총 12종의 PFCs를 분석하였으며, PFBS를 제외한 PFCs의 회수율은 82.2-119.4%였으며, 정밀도는 2.2-28.9%였다. PFCs물질별로는 PFPeA(84.1%)와 PFOA (82.8%) 검출률이 높았고, PFOS의 검출률은 29.1%였으며, 대부분의 시료에서 PFOA가 PFOS보다 잔류 수준이 높은 경향을 보였다. 시료의 총 PFCs의 평균 농도는 1.68±1.79 ng/g이었으며, PFPeA와 PFOA의 비중이 높았다. Perfluorinated compounds (PFCs) are linked to adverse effect on human healths, therefore, the aim of this study was to determine the levels of PFCs in prepared food. One hundred and fifty-one samples of six prepared food groups were purchased from retail food establishments in Korea. Twelve PFCs were detected in the sample, the accuracy ranged between 80.8-119.4% and precision ranged between 2.2-2.89% high and low concentrations in PFCs spiked samples. The detection frequency of perfluoro-pentanoic acid (PFPeA) was and perfluorooctanoic acid (PFOA) above the limit of detection, which was 84.1 and 82.8%, respectively, but positive detection frequency of perfluorooctanesulphonic acid (PFOS) was as shown as low as 29.1%. Residual PFOA levels in most of the samples were higher than the PFOS levels. The average concentration of total PFCs in the samples was 1.68±1.79 ng/g, and PFPeA and PFOA were the predominant PFCs.

서비스직 근로자들의 영수증 접촉 정도에 따른 비스페놀A 노출량 조사

이유나,이유진,장지원,한가을,고영림,Lee, Yuna,Lee, Yujin,Jang, Jiwon,Han, Gaeul,Kho, Younglim 한국환경보건학회 2014 한국환경보건학회지 Vol.40 No.6

Objectives: Bisphenol A, or BPA, is a chemical component in polycarbonate plastic with which many people come into contact every day. A great deal of controversy has arisen over its safety since this material, which is known to disrupt the human endocrine system and cause neurological difficulties and cancer, is commonplace in beverage containers, food can liners, and receipt paper rolls. In this study, we determined the levels of exposure to BPA of workers in the service industry depending on the number of receipts contacted. Methods: The participants were 16 male and 18 female workers employed in the service industry. Using a questionnaire, we investigated general and job characteristics. Urine samples were collected and analyzed by the LC-MS/MS technique after enzymatic hydrolysis and solid phase extraction (SPE). Results: The geometric mean (GM) concentration of urinary BPA from all subjects was 1.02 ng/ml. Workers were exposed significantly to more BPA according to the number of receipts they contacted, their work experience, and working hours per day. The BPA concentration of those who touched more than 100 receipts per day was 3.09 ng/ml, while that of the other participants was 0.61 ng/ml. It was shown that wearing gloves can protect from BPA exposure. Conclusion: We determined the urinary BPA concentrations of workers in service industry and found that the contact with receipts could increase the BPA exposure of service workers.

세척제 잔류량 확인을 위한 계면활성제 분석법 확립

박나연,이소정,김정환,고영림,Park, Na-Youn,Lee, Sojeong,Kim, Jung Hoan,Kho, Younglim 대한화학회 2021 대한화학회지 Vol.65 No.6

계면활성제는 하나의 분자에 친수성 작용기와 소수성 부분이 동시에 존재하는 유기화합물로 수용액 상태에서 전하에 따라 비이온, 음이온, 양이온, 양쪽성 계면활성제로 분류된다. 1종 세척제(야채 및 과일 세척제)를 사용하여 야채나 과일을 세척할 때 극미량이 잔류하여 섭취를 통해 인체에 노출될 가능성이 있는 것으로 알려져 있다. 본 연구는 세척제를 사용한 야채, 과일의 세척 후 세척제 잔류량 분석을 위해 LC-MS/MS를 이용한 계면활성제 5종 동시분석법을 확립하였다. 이동상으로는 50 mM 암모늄 포메이트와 0.1% 폼산이 함유된 증류수와 아세토니트릴을 사용하였으며, XBridge BEH C8 컬럼을 이용하여 경사용매조성법으로 분석하였다. 확립된 분석법의 정확도는 83.9~112.1% 이었고, 정밀도는 20% 이하로 정밀한 값을 보였다. 검출한계는 7.0(SLS)~29.0(SLES-N3) ㎍/L이었고, 검량선 회귀식의 상관계수(r²)은 0.99 이상으로 우수한 직선성을 보여주어 야채 및 과일에 잔류하는 미량의 계면활성제 성분의 분석에 적합한 것으로 판단된다. Surfactants are organic compounds that have both hydrophilic and non-polar parts in one molecule, classified as non-ion, anion, cation, and amphoteric surfactants according to the charge of hydrophilic parts in aqueous state. A trace amounts may remain when vegetables and fruits are washed using type1 detergent (Vegetable and fruit detergent), and there is a possibility of exposure to the human body through ingestion. This study developed the simultaneous analysis method for 5 surfactants with LC-MS/MS for analysis of detergent residues after washing vegetables and fruits with detergent. The mobile phase used distilled water and acetonitrile containing 50 mM ammonium formate and 0.1% formic acid and was analyzed using a gradient method using XBridge BEH C8 column. The accuracy of the established method was 83.9-112.1%, and the precision was less than 20%. The detection limit was 7.0 (SLS) to 29.0 (SLES-N3) ㎍/L, and the correlation coefficient (r²) of calibration line regression was greater than 0.99, it is considered suitable for the analysis of trace amounts of surfactant components remaining in vegetables and fruits.

곶자왈 지역의 의약물질과 농약의 오염수준과 생태독성영향

강하병,고수림,최윤송,이상우,고영림,오달영,최경호,Kang, Ha Byeong,Koh, Su Rim,Choi, Yoonsong,Lee, Sangwoo,Kho, YoungLim,Oh, Dalyoung,Choi, Kyungho 한국환경보건학회 2013 한국환경보건학회지 Vol.39 No.5

Objective: Gotjawal refers to a special geographical designation found in Jeju Province, Korea, where vegetation forms over a rocky area. Due to the important ecological value of Gotjawal, international concern about such areas is growing. However, only limited information is available regarding environmental contamination of Gotjawal. This study was conducted in order to investigate the levels of contamination and associated ecotoxicological effects of surface water bodies in Gotjawal. Methods: Surface water samples were collected at three sampling sites in two Gotjawal areas and were analyzed for several pharmaceuticals and pesticides in consideration of the potential sources of contamination. The ecotoxicity of the samples was measured using water fleas (Daphnia magna and Moina macrocopa) and zebrafish (Danio rerio). In addition, effects on the gene transcription of zebrafish were investigated following exposure to the samples. Results: Nine pharmaceuticals were detected in the samples, but none of the target pesticides were detected. Following acute exposure to two surface water samples, the survival of waterfleas was significantly reduced, but reproduction was not affected. In zebrafish, time-to-hatch was delayed and fry survival was decreased in some samples. On the other hand, at the transcriptional level, there were no genes significantly influenced by exposure to the samples. Conclusion: This is the first study investigating environmental contamination in Gotjawal areas and associated ecotoxicities. Further studies are warranted to identify the cause of acute Daphnia toxicity and to determine potential consequences of longer-term exposure in Gotjawal areas.

LC-MS/MS를 이용한 동물 사료 내 글라이포세이트 및 글루포시네이트 분석

이지수,김완서,양희득,박나연,정웅,김정환,고영림,Lee, Ji-Su,Kim, Wanseo,Yang, Heedeuk,Park, Na-Youn,Jung, Woong,Kim, Junghoan,Kho, Younglim 대한화학회 2019 대한화학회지 Vol.63 No.5

식품에서 글라이포세이트와 글루포시네이트의 함량 기준은 구체적이며 분류가 잘 되어있다. 그러나 동물 사료나 먹이에서의 함량 기준은 비교적 구체적이지 못하며 분류가 미흡한 편이다. 또한 인간에게 미치는 위험에 대해서는 지속적인 논의는 이루어지고 있는 반면에, 동물에게 미치는 위험성은 충분한 연구가 이루어지지 않았다. 본 연구에서는 동물이 글라이포세이트를 섭취하는 경로로 추정되는 사료에서의 분석법을 확립하였다. 25% 메탄올을 이용한 용매추출, 원심분리 후 고체상추출로 정제한 후, 농축하여 LC-MS/MS를 이용하여 정량 분석하였다. 분석법 검증은 검출한계, 정확도 및 정밀도 시험을 통해 진행하였다. 확립된 시험법의 검출한계는 글루포시네이트 $1.8{\mu}g/kg$, 글라이포세이트 $2.4{\mu}g/kg$ 이었다. 정확도는 94.4~103.4%, 정밀도는 1.5~7.2%의 범위로 나타났다. 동물 사료(n=13)를 분석법을 적용시켜 분석한 결과 글루포시네이트는 한 개의 시료에서 검출되었고($ND{\sim}8.8{\mu}g/kg$), 글라이포세이트는 한 개의 시료를 제외한 모든 시료에서 검출($ND{\sim}337.0{\mu}g/kg$)되었다. The standards for the contents of glyphosate and glufosinate in foods are specific and well categorized. However, the standard of content in animal feeds is relatively inadequate and the classification is insufficient. There is also constant debate about the risk of glyphosate and glufosinate to human health, but the risk to animals has not been well studied. In this study, we established an analytical method in feeds that is estimated to be the path for animals to ingest glyphosate. The solvent extraction was carried out using 25% methanol. After centrifugation, samples were purified using solid phase extraction (SPE) and quantitatively analysed using LC-MS/MS after concentrated. Assessment of validation was conducted through detection limits, accuracy, and precision tests. The detection limits for the established method were 1.8 of ${\mu}g/kg$ of glufosinate and $2.4{\mu}g/kg$ of glyphosate. Accuracy was ranged from 94.4% to 103.4% and precision was range from 1.5% to 7.2%. Glufosinate was detected in one sample ($ND{\sim}8.8{\mu}g/kg$) and glyphosate was detected in all but one sample ($ND{\sim}337.0{\mu}g/kg$) by applying the analytical method to animal feeds (n=13).

소변 중 다환방향족탄화수소 대사체의 분석법 확립 및 교차분석

박나연,전중대,구혜령,김정환,이은희,이경무,문철진,고영림,Park, Na-Youn,Jeon, Jung-Dae,Koo, Hyeryeong,Kim, Jung Hoan,Lee, Eun-Hee,Lee, Kyungmu,Mun, Cheoljin,Kho, Younglim 한국환경보건학회 2015 한국환경보건학회지 Vol.41 No.5

Objectives: This study was performed to evaluate the analytical method for PAH metabolites in human urine using enzyme hydrolysis and solid-phase extraction coupled with LC-(ESI)-MS/MS technique. Methods: We employed HPLC tandem mass spectrometry techniques with appropriate pre-treatment for analysis of 16 OH-PAHs in human urine. Samples were hydrolysis by ${\beta}$-flucuronidase/Aryl sulfatase, and target compounds were extracted by solid-phase extraction with a strata-x cartridge. Cross-validation was performed between Eulji University and Green Cross laboratories with 200 human urine samples. Results: The accuracies were between 90.3% and 118.8%, and precisions (relative standard deviations) were lower than 10%. The linearity obtained was satisfying for the 16 OH-PAH compounds, with a coefficient of determination ($r^2$) higher than 0.99. The results of cross-validation at the two organizations were compared by ICC (interclass correlation coefficient) values. The cross-validation results were excellent or good for all compounds. Conclusion: An analytical method was validated for low nanogram levels of 16 OH-PAHs in human urine. Also, satisfying results were obtained for method validation such as accuracy, precision and ICC of cross-validation.

대학생들의 화장습관에 따른 소변 중 벤조페논 농도 비교

오승은,호성욱,김현석,이세훈,박나연,고영림,Oh, Seungeun,Ho, Sungwook,Kim, Hyunsuk,Lee, Sehoon,Park, Nayeon,Kho, Younglim 한국환경보건학회 2013 한국환경보건학회지 Vol.39 No.6

Objectives: Ultraviolet (UV) radiation may cause skin cancer, photo-ageing, erythema, and sunburn. Benzophenone (BP) is commonly used to protect skin from UV radiation. In Korea, sunscreen, sunblock, socalled 'blemish balm' (BB) and 'color correcting' (CC) creams, and foundation may contain from 0.5 to 5% benzophenone in order to protect skin from UV radiation. The purpose of this study is to understand the levels of benzophenone derivatives in urine among a group of university students and identify the contribution of cosmetics use. Methods: Forty volunteers (20 women and 20 men) were asked about skin type, frequency of use of cosmetics, and recognition of related health effects, etc. in a survey. Subjects were divided into several subgroups and were compared for concentration of benzophenone-1 (BP-1) and benzophenone-3 (BP-3). Their urine was pretreated with enzyme hydrolysis and solid phase extraction. Determinations of BP-1 and BP-3 in the urine were made with LC-MS/MS. Results: Among the study subjects, 82.5% used basic cosmetics at least once per day, and 77.5% used sunscreens at least once per day. The concentrations of BP-1 and BP-3 of the males were 4.36 ng/mL and 9.16 ng/mL, respectively. Those of the females were 3.98 ng/mL and 5.07 ng/mL, respectively. The use of cosmetics was positively related to urinary benzophenone levels. Conclusions: BP-1 and BP-3 were widely detected among the university students. Cosmetic use was identified as a potential source. Implications of such exposure deserve further investigation.