식품에서 당살초 판별을 위한 LC-ESI-MS/MS 분석법과 KASP 마커 개발

박보름,이선희,엄권용,노은영,한경문,황진우,김형일,백선영 한국식품위생안전성학회 2022 한국식품위생안전성학회지 Vol.37 No.2

Known for its effectiveness in weight loss and diabetes prevention, Gymnema sylvestre products can be found in the US, Japanese, and Indian markets. However, the recommended dosage or safety of these products has not yet been proven. Therefore, development of an analytical method for detecting the content of Gymnema sylvestre in food products is required. Accordingly, this study proposes an analysis method that can examine Gymnema sylvestre in food using LC-ESI-MS/MS and KASP (Kompetitive Allele-Specific PCR) markers. In LC-ESI-MS/MS, a simultaneous analysis method for gymnemic acid and deacylgymnemic acid was optimized using negative ionization mode, and its validation test was completed for solid and liquid samples. In addition, KASP markers were prepared by finding the specific SNP of G. sylvestre in ITS2 and matK through DNA barcodes. The two KASP markers returned positive FAM fluorescence result when combined with G. sylvestre, and this aspect was confirmed in raw G. sylvestre as well. The applicability of the method was tested on 21 different food and healthy functional products containing G. sylvestre purchased on the internet. As a result, although there was a difference in the ratios of gymnemic acid and deacylgymnemic acid in LC-ESI-MS/MS, the index component was detected in all 21 products samples. In the KASP analysis, 9 products returned positive FAM result, and the rest of the products were found to be containing G. sylvestre extract. This study is the first study to use the dual system of LC-ESI-MS/MS and KASP for the analysis of G. sylvestre. The study has confirmed that these two methods are applicable to the examine G. sylvestre content in food products.

나문재 추출물의 성분 분석

양희정 ( Hee Jung Yang ),박수남 ( Soo Nam Park ) 대한화장품학회 2008 대한화장품학회지 Vol.34 No.3

이전 연구에서 저자들은 나문재 추출물의 항산화 작용과 추출물 함유 크림의 유화 안정성에 대한 결과를 보고한 바 있다[1,2]. 본 연구에서는 thin layer chromatography (TLC), high performance liquid chromatography (HPLC)와 liquid chromatography/Electrospray Tandem mass spectrometry (LC/ESI-MS/MS), ¹H-NMR을 이용하여 나문재 추출물에 대한 성분 분석을 수행하였다. 나문재 추출물 중 ethyl acetate 분획의 TLC는 5개의 띠(SA 1 ~ SA 5)로 분리되었다. Ethyl acetate 분획의 당 제거반응 후 얻어진 aglycone 분획에 대한 HPLC 크로마토그램은 2개의 피이크(SAA 2 및 SAA 1)를 나타냈고, 각각 그 용리 순서는 quercetin, kaempferol이었으며 조성비는 quercetin 16.88 %, kaempferol 83.12 %로 kaempferol의 함량이 큰 것으로 나타났다. 또한 LC/ESI-MS/MS를 통해서 SA 2는 kaempferol-3-O-glucoside로 SA 3는 quercetin-3-O-glucoside, SA 4는 kaempferol-3-O-rutinoside, SA 5는 quercetin-3-O-rutinoside로 확인되었다. LC/ESI-MS/MS의 스펙트럼에서 SAA 1은 탈양성자화된 aglycone 분획에 상응하는 분자이온 [M-H]-(m/z 285) 피이크를 나타냈으며, ¹H-NMR 분석을 실시한 결과 [δ 6.19 (1H, d, J = 1.8 Hz, H-6), δ 6.44 (1H, d, J = 1.8 Hz, H-8), δ 6.92 (2H, d, J = 9.0 Hz, H-3',5'), δ 8.04 (2H, d, J = 9.0 Hz, H-2',6')]에서 피이크들이 나타났다, 따라서 SAA 1은 kaempferol임이 확인되었다. SAA 2는 aglycone 분획에 상응하는 분자이온 [M-H]- (m/z 301)을 생성하였고, ¹H-NMR 스펙트럼은 [δ 6.20 (1H, d, J = 2.0 Hz, H-6), δ 6.42 (1H, d, J = 2.0 Hz, H-8), δ 6.90 (1H, d, J = 8.6 Hz, H-5'), δ 7.55 (1H, dd, J = 8.6, 2.2 Hz, H-6'), δ 7.69 (1H, d, J = 2.2 Hz, H-2')]에서 피이크들을 나타냈고, 따라서 SAA 2는 quercetin으로 확인되었다. 결론적으로, 이미 보고된 나문재 추출물의 항산화 작용 그리고 안정성 실험과 더불어 나문재 추출물의 성분 분석은 새로운 기능성 화장품원료로서 응용이 가능함을 시사한다. In the previous study, the anti-oxidant activity of extract/fraction of Sueada aspparagoides (SA) and the stability test for the cream containing SA extract were investigated respectively[1,2]. In this study, the components of SA extract were analyzed by TLC, HPLC, and LC/ESI-MS/MS, ¹H-NMR. TLC chromatogram of ethyl acetate fraction of SA extract revealed 5 bands (SA 1 ∼ SA 5). HPLC chromatogram of aglycone fractions obtained from deglycoylation reaction of ethyl acetate fraction showed 2 bands (SAA 2 and SAA 1), which were identified as quercetin (composition ratio, 16.88 %) and kaempferol (83.12 %) in the order of elution time. Among 5 bands of TLC chromatogram, 4 bands (SA 2 ∼ SA 5) also were identified as kaempferol-3-O-glucoside (SA 2), quercetin-3-O-glucoside (SA 3), kaempferol-3-O-rutinoside (SA 4), quercetin-3-O-rutinoside (SA 5) by LC/ESI-MS/MSMS/MS, respectively. The spectrum generated for SAA 1 by LC/ESI-MS/MS in the negative ion mode also gave the ion corresponding to the deprotonated aglycone [M-H]- (285 m/z), the ¹H-NMR spectrum contained signals [δ 6.19 (1H, d, J = 1.8 Hz, H-6), δ 6.44 (1H, d, J = 1.8 Hz, H-8), δ 6.92 (2H, d, J = 9.0 Hz, H-3',5'), δ 8.04 (2H, d, J = 9.0 Hz, H-2',6')], thus SAA 1 was identified as kaempferol. SAA 2 yielded the deprotonated agycone ion [M-H]- (301 m/z), ¹H-NMR spectrum showed signals [δ 6.20 (1H, d, J = 2.0 Hz, H-6), δ 6.42 (1H, d, J = 2.0 Hz, H-8), δ 6.90 (1H, d, J = 8.6 Hz, H-5'), δ 7.55 (1H, dd, J = 8.6, 2.2 Hz, H-6'), δ 7.69 (1H, d, J = 2.2 Hz, H-2')], thus SAA 2 was identified as quercetin. In conclusion, with the anti-oxidant activity and the stability test reported previously, component analysis of SA extracts could be applicable to new cosmeceuticals.

Simultaneous determination of five coumarins in Angelicae dahuricae Radix by HPLC/UV and LC-ESI-MS/MS

Park, Ah Yeon,Park, So-Young,Lee, Jaehyun,Jung, Mihye,Kim, Jinwoong,Kang, Sam Sik,Youm, Jeong-Rok,Han, Sang Beom John Wiley Sons, Ltd. 2009 Biomedical chromatography Vol.23 No.10

<P>Rapid, simple and reliable HPLC/UV and LC-ESI-MS/MS methods for the simultaneous determination of five active coumarins of Angelicae dahuricae Radix, byakangelicol (1), oxypeucedanin (2), imperatorin (3), phellopterin (4) and isoimperatorin (5) were developed and validated. The separation condition for HPLC/UV was optimized using a Develosil RPAQUEOUS C<SUB>30</SUB> column using 70% acetonitrile in water as the mobile phase. This HPLC/UV method was successful for providing the baseline separation of the five coumarins with no interfering peaks detected in the 70% ethanol extract of Angelicae dahuricae Radix. The specific determination of the five coumarins was also accomplished by a triple quadrupole tandem mass spectrometer equipped with an electrospray ionization source (LC-ESI-MS/MS). Multiple reaction monitoring (MRM) in the positive mode was used to enhance the selectivity of detection. The LC-ESI-MS/MS methods were successfully applied for the determination of the five major coumarins in Angelicae dahuricae Radix. These HPLC/UV and LC-ESI-MS/MS methods were validated in terms of recovery, linearity, accuracy and precision (intra- and inter-day validation). Taken together, the shorter analysis time involved makes these HPLC/UV and LC-ESI-MS/MS methods valuable for the commercial quality control of Angelicae dahuricae Radix extracts and its pharmaceutical preparations. Copyright © 2009 John Wiley & Sons, Ltd.</P>

LC-ESI-MS에 의한 사군자탕의 지표성분 분석

서창섭,신현규 한국생약학회 2019 생약학회지 Vol.50 No.1

One of the oriental medicine prescriptions, Sagunja-tang consists of four herbal medicines (Ginseng Radix, Poria Sclerotium, Atractylodis Rhizoma Alba, and Glycyrrhiziae Radix et Rhizoma) and has been used as a medicine to enhance tonify the function of spleen and stomach in Korea. In this study, we conducted simultaneous analysis of the 9 marker components, liquiritin apioside, liquiritin, ginsenoside Rg1, liquiritigenin, ginsenoside Rb1, glycyrrhizin, atractylenolide III, atractylenolide II, and atractylenolide I in Sagunja-tang using a liquid chromatography–electrospray ionization-mass spectrometry (LC-ESI-MS/MS). Marker compounds were separated on a Waters Acquity UPLC BEH C18 analytical column (2.1 × 100 mm, 1.7 mm) and the column was maintained at 45oC. The mobile phase consists of 0.1% (v/v) aqueous formic acid and acetonitrile with gradient condition. The LC-MS analysis was performed using a Waters ACQUITY TQD LC-MS/MS system with multiple reaction monitoring (MRM) method in the positive and negative modes. The calibration curves of the nine marker components showed good linearity with coefficient of determination ³ 0.9984 within tested range. The limits of detection and limits of quantification values were 0.27–2.42 ng/mL and 0.81–7.27 ng/mL, respectively. The concentrations of tested 9 analytes in the lyophilized Sagunja-tang sample using the established LC-ESI-MS/MS MRM method were detected up to 16.593 mg/g. These results can be useful as a basic data for the quality control of an oriental medicine prescriptions.

LC-ESI-tandem MS를 이용한 기능성표방식품 중 부정유해물질 신속검사체계 개발

김희연(Hee-Yun Kim),이화미(Hwa-Mi Lee),장영미(Young-Mi Jang),주현진(HyunJin Joo),정용현(Young-Hyun Jung),이명숙(Myoung-Sook Lee),박종석(Jong-Seok Park),이광호(Kwang-Ho Lee) 한국식품과학회 2007 한국식품과학회지 Vol.39 No.4

부정유해물질 총 13종에 대한 신속하고 고감도의 LC-ESI-MS-MS 동시분석 방법을 개발하였으며 바데나필을 포함한 11종은 ESI positive 모드에서 타다라필을 포함한 2종은 ESI negative 모드에서 검출하는 방법으로서 시료 전처리는 간단히 메탄올 추출법을 사용하였다. 기능성표방식품 중 부정유해물질의 확인은 한번 시료를 주입함으로써 15분 이내에 13종의 분석이 가능하고 크로마토그램의 분리는 아세토니트릴과 10 mM ammonium formate가 들어있는 탈이온수를 이용한(pH 7.0) 기울기 용매 조건으로 수행하였다. 확립한 13종에 대한 부정유해물질 분석방법은 검출한계(LOD)는 0.1-5ng/㎖이고, 정량한계(LOQ)는 0.1-10ng/㎖으로서 평균 상관계수(r²)는 0.9853로서 ppb 수준에서 정량성을 가지며 회수융은 87.5-98.5%, 변동계수는 15% 이하임을 확인할 수 있었다. 또한 확립한 시험방법 LC/MS/MS를 이용하여 147건의 기능성표방식품 중의 부정유해물질의 검증을 실시한 결과, 유해 물질의 검출이 나타타지 않음을 알 수 있었다. 기능성표방식풍중의 실데나필과 그 유사물질을 포함한 13종의 부정유해물질에 대한 스크리닝 방법으로 MRM 모드를 이용한 LC-ESI-MS-MS 방법을 개발하였으며, 이는 유해물질에 대한 고성능액체크로마토그래피/자외선흡광광도법의 선택성등의 제한성을 극복한 부정유해물질의 스크리닝에 신속하고 미량까지 검출 가능한 가치 있는 방법임을 확인할 수 있었다. A high-performance liquid chromatography-electrospray ionization (HPLC-ESI) tandem MS was developed for the rapid and simultaneous determination of forbidden medicines in dietary supplements. Thirteen medicinal components such as PDE-5 inhibitors and their analogues, and the newly identified dimethylsildenafil and xanthoanthrafil, were included in this study. After tentative standardization of molecular ions in both polarities using thirteen references on the mass spectrometer, with ESI-continuous infusion via the syringe pump method, the relative intensity of the ions present in the resulting spectra was quantitatively compared. From the results, the ion mode was selected depending on each reference’s characteristics. A HPLC method coupled with the ESI mode was developed considering the matrix effect and interference depending on the type of sample. The validation test of the developed method was followed by carrying out precision, accuracy, recovery, sensitivity and linearity, etc. The method showed sufficiently high sensitivity, reproducibility, and specificity, and produced 4 times faster results when compared with the existing HPLC/UV method for the determination of forbidden compounds in dietary supplements.

LC-ESI-MS/MS를 이용한 계지탕 중 주요 성분 분석

서창섭,하혜경 한국생약학회 2018 생약학회지 Vol.49 No.1

A traditional herbal formula, Gyeji-tang has been used to treat the early colds, headache, chills, and fever in Asian countries. In this study, we were performed simultaneous determination of the 14 bioactive marker compounds, gallic acid, spinosin, paeoniflorin, albiflorin, liquiritin apioside, liquiritin, 6ʹʹʹ-feruloylspinosin, liquilitigenin, coumarin, cinnmamic acid, benzoylpaeoniflorin, cinnamaldehyde, glycyrrhizin, and 6-gingerol in Gyeji-tang using an ultra–performance liquid chromatography–electrospray ionization-mass spectrometry (UPLC-ESI-MS/MS). Analytical column was used a Waters Acquity UPLC BEH C18 analytical column (2.1×100 mm, 1.7 μm) and maintained at 45oC with a flow rate of 0.3 mL/min. The mobile phase consists of 0.1% (v/v) formic acid in water and acetonitrile with gradient elution. The MS analysis was conducted using multiple reaction monitoring in the positive and negative modes by a Waters ACQUITY TQD LC-MS/MS system. The calibration curves of 14 bioactive marker compounds showed linearity with correlation coefficients ³ 0.9798. The limits of detection and quantification values were in the range of 0.11-6.66 ng/mL and 0.34-19.99 ng/mL, respectively. As a result of the analysis using the established LC-MS/MS method, the amounts of tested 14 compounds in the lyophilized Gyeji-tang sample were detected up to 85.7 μg/g. These results may be useful for quality assessment of a traditional herbal formulas.

LC-ESI-MS/MS를 이용한 평위산 주요 성분의 함량 분석

서창섭,신현규 한국생약학회 2018 생약학회지 Vol.49 No.3

Pyungwi-san has been used to treat the digestive system diseases, physconia, nausea, anorexia, and dyspepsia in Korea. In this study, an ultra–performance liquid chromatography–electrospray ionization-mass spectrometry (UPLC-ESI-MS/MS) method was optimized for simultaneous determination of the 14 marker components, spinosin, liquiritirn apioside, liquiritin, narirutin, 6ʹʹʹ-feruloylspinosin, hesperidin, liquiritigenin, glycyrrhizin, 6-gingerol, atractylenolide III, honokiol, atractylenolide II, magnolol, and atractylenolide I in Pyungwi-san extract. All analytes were separated on a Waters Acquity UPLC BEH C18 analytical column (2.1×100 mm, 1.7 μm) with maintained at 45oC. The mobile phase consisted of 0.1% (v/v) aqueous formic acid and acetonitrile. The MS conditions were as follows: capillary voltage 3.3 kV, extractor voltage 3.0 V, RF lens voltage 0.3 V, source temperature 120oC, desolvation temperature 300oC, desolvation gas 600 L/h, cone gas 50 L/h and collision gas 0.14 mL/min. The coefficient of determination of 14 analytes was 0.9989–1.0000. The limits of detection and quantification values of the all analytes were 0.04–2.56 and 0.13–7.69 ng/mL, respectively. As a result of the analysis using the established LC-ESI-MS/MS method, the 5 components, spinosin, 6'''-feruloylspinosin, atractylenolide III, II, and I derived from Zizyphi Fructus and Atractylodis Rhizoma, were not detected in this extract. On the other hand, the 9 components except for the 5 components were 4.15–498.87 mg/kg in lyophilized Pyungwi-san extract. Among these components, glycyrrhizin, marker compound of Glycyrrhizae Radix et Rhizoma, was detected the most amount as a 498.87 mg/kg.

LC-ESI-MS/MS를 이용한 생체시료 중 브롬화피나베리움의 고감도 분석 및 이를 이용한 생체이용률 평가 : Applicability to Oral Bioavailability Determination

박석,이예리,김호현,이희주,김윤균,염정록,한상범 한국약제학회 2004 Journal of Pharmaceutical Investigation Vol.34 No.6

A sensitive method for quantification of pinaverium bromide in human plasma was established using liquid chromatography-electrospray ionization tandem mass spectrometrv(LC-ESI-MS/MS). Glimepiride was used as internal standard. Pinaverium bromide and internal standard in plasma sample were extracted using tert-but}lmethvlether(TBME). A centrifuged upper laver was then evaporated and reconstituted with mobile phase of acetonitrile-5 m1VI ammonium formate (8020. pH 3.0). The reconstituted samples were injected into a C_(18) reversed-phase column. Using MS/MS with multiple reaction monitoring (MRM) mode. pinaverium and glimepirde were detected without severe interference from human plasma matrix. Pinaverium produced a protonated precursor ion ([M+H]^(+)) at m/z 510.3 and a corresponding product ion at m/z 228.9. Internal standard produced a protonated precursor ion ([M+H] ^(+)) at m/z 491.5 and a corresponding product ion at m/z 352.0. Detection of pinaverium bromide in human plasma was accurate and precise. with limit of quantitation at 0.5 ng/ml. The method has been successfully applied to bioavailability study of pinaverium bromide tablet in Korean healthy male volunteers. Pharmacokinetic parameters such as AUCr. C_(max) T_(max). K_(el) and t_(l/2) were calculated.

Rapid identification and quantification of bioactive metabolites in processed Camellia sinensis samples by UHPLC-ESI-MS/MS and evaluation of their antioxidant activity

Naheed Akhtar,Vinitha Moolchand Thadhani,Faraz Ul Haq,Muhammad Noman Khan,Sajjad Ali,Syed Ghulam Musharraf 한국공업화학회 2020 Journal of Industrial and Engineering Chemistry Vol.90 No.-

Tea beverages have been enjoyed globally for the last several decades. The present study focuses on the comprehensive chemical and biological analysis of three processed tea products. A total of sixty-three compounds in processed samples were identified based on their exact masses and fragmentation patterns using LC-ESI-QTOF-MS/MS. Furthermore, quantification of eight analytes including caffeine (1), theophylline (2), (+)-catechin (3), (-)-epicatechin (4), (-)-epicatechin gallate (5), (-)-gallocatechin (6), (-)-epigallocatechin gallate (7) and quercetin-3-d-β-glucoside (8) in tea samples was performed using LC-ESI-IT-MS/MS. The concentrations of analytes were found in the range of 0.03 mg/g to 75.58 mg/g in all tea samples. The developed method showed excellent accuracy as %bias ranged from 0.2–3.69% and a good precision with %RSD ranged from 0.03 to 5.11. The LOD and LOQ for all the analytes were found to be in the range of 0.16–3.26 ng/mL and 0.44–9.87 ng/mL, respectively. The DPPH scavenging effects of samples were also investigated and all the samples were found to be strong scavengers of DPPH radical showing 69.50 ± 0.01–78.60 ± 0.10 %RSA. The established method provides a useful way for understanding the metabolite distribution in processed products of C. sinensis and to develop quality control protocols for these products.

LC/ESI-MS/MS를 이용한 축산폐수처리장의 의약물질 모니터링

서천규 ( Cheon Kyu Seo ),김병주 ( Byung Ju Kim ),조현우 ( Hyun Woo Cho ),남윤구 ( Youn Ku Nam ),김탁현 ( Tak Hyun Kim ),이면주 ( Myun Joo Lee ),명승운 ( Seung Woon Myung ) 한국환경분석학회 2009 환경분석과 독성보건 Vol.12 No.4

In this study, an effective monitoring and the investigation of treatment efficiency of pharmaceuticals from the influent and effluent of livestock wastewater treatment plants (WWTPs) by LC/ESI-MS/MS was performed. Thirteen pharmaceuticals including anntibiotics, growth promoters and disinfectants were assayed from twelve WWTPs in South Korea. The analytical method for pharmaceuticals from wastewater samples gave above 60% of recoveries from the spiked blank samples. The calibration curves showed good linearities (above r2=0.99) in the concentration range of 0.01~10.00 ng/mL. Limits of detection (LOD, at S/N>3) were the range of 0.00128~0.04505 ng/mL, and methods detection limits (MDL, at RSD<20% and S/N>10) were 0.00552~0.15015 ng/mL in surface water. Accuracy and precision from spiked blank samples were -12.4~20.6% and 0.6~24.0% from spiked blank samples, respectively. The established method could be used to determine low concentration levels of pharmaceuticals in environmental samples. From few influents of livestock WWTPs, chlortetracycline and acetaminophen were detected with the highest concentration (70.87 μg/ mL and 1.35 μg/mL, respectively) among the monitoring pharmaceuticals. And also lincomycin, sulfathiazole, sulfamethazine, trimethoprim, acetyl salicylic acid, tylosin, glutaraldehyde and formaldehyde were detected with 0.21~294.02 ng/mL from the influents of WWTPs.