1. Mass Spectroscopy 의기본원리 원자및분자를기체상 (high-vacuum region) 에서이온화시켜하전입자 ( 분자또는원자 ) 에대한질량 (mass/charge) 을측정하여 molecular weight을결정하는방법, 더나아가복잡한구조를가진화합물의구조

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1 의약외품 화장품품질확보를위한워크숍 GC/MS 및 LC/MS 의이해및응용 Matrix-Assisted Laser Desorption Ionization Timeof-Flight (MALDI- TOF) Electrospray Ionizationquadrupole-Timeof-Flight tandem MS (ESI-q-TOF tandem MS) Liquid Chromatography- Mass Spectrometry (LC-MS) Gas Chromatography- Mass Spectrometry (GC-MS) 이화여자대학교권영주

2 1. Mass Spectroscopy 의기본원리 원자및분자를기체상 (high-vacuum region) 에서이온화시켜하전입자 ( 분자또는원자 ) 에대한질량 (mass/charge) 을측정하여 molecular weight을결정하는방법, 더나아가복잡한구조를가진화합물의구조를규명할수있는방법 시료도입부이온화부이온분리부이온검출부이온기록부 Ionization source Mass analyzer Ion detector Sample inlet Data system Atmosphere / vacuum High vacuum Figure 1 질량분석기의구성도 Function1. 물질을 high-energy electron 으로공격하여이온화시키고 electric field 에서가속화시킴 : ionization chamber Function2. 가속화된이온은 magnetic or electric field 에서그것의 molecular-tocharge ratio 에따라분리. Function3. 특정 molecular-to-charge ratio 를가지는이온을 detector 에서확인.

3 분자의이온화및조각화과정 Most of the instrument is designed to detect cations. most fragments have a charge of +1, the M +. usually represents the molecular weight of the fragment. spectrum: relative abundance (signal intensity) vs m/z

4 화합물의 mass spectrum 예 (m/z)

5 2. Mass Spectroscopy 의특성및활용 MS는분리분석법인기체및액체크로마토그래피와의결합이용이하여혼합물의분리, 구조확인및극미량분석에있어서매우유용한수단이다. 유기물의극미량분석에서 GC-MS는다른분석장비에비하여감도 (sensitivity) 가월등히우수하다. 1990년대이후부터연이온화법인고속원자폭격법 (fast atom bombardment, FAB), 전자분무이온화법 (electrospray ionization, ESI), 매트릭스보조레이저탈착이온화법 (matrix-assisted laser desorption ionization, MALDI) 등이개발되면서복잡한유기화합물의구조분석이급격하게발전을이루게되어최근에는최첨단분석법으로발전하게되었다. 환경분석 : 독성이강한다이옥신과같은극미량 (picogram level, g) 및고난이도분석이질량분석기로가능 고분자물질인단백질, DNA으로부터저분자물질대사체에이르기까지다양한생체물질의구조분석에활용되고있다. 아폴로우주선에서달의지표성분의확인, 올림픽경기에서도핑테스트 ( 금지약물의분석 ), 불법마약류의검출, 약물대사연구, 임상연구, 신약개발, 환경오염물질검출및생체고분자물질의구조규명등다양한과학분야에질량분석법이적용

6 3. Ionization Method v 일반적인방법 (standard method): EI, CI v 연이온화법 (soft ionization): 탈리화학이온화법 (Desorption Chemical Ionization, DCI), 이차이온화 (Secondary ion, SIMS) 과고속원자폭격 (Fast atom bombardment, FAB), 매트릭스보조레이저탈착이온화 (Matrix-assisted laser desorption ionization, MALDI), 장이온화법 (Field ionization, FI) 과장탈착법 (Field desorption, FD), Electrospray Ionization (ESI) 대기압이온화 (Atmospheric pressure ionization, API)

7 A. Electron Ionization (EI) 저분자유기화합물이온화에가장널리사용되고주로양이온을생성 기체상태의시료분자를전류로가열한필라멘트로부터방출되는전자빔 (70 ev) 으로충돌하여이온화시킴 과잉이온화에너지 ( 이온화전위 ) 를주는이유는구조정보에도움을주는조각이온을얻을수있고전압이약간변해도이온의생성확율이별로변하지않는장점이있기때문임. 전자빔을에너지 à 전자 1 개가방출 à 분자이온 (molecular ion, M + 또는 M + ) 생성 à 여분의에너지를가진 M + 는결합의약한부분이끊어져조각이온 (fragment ion) 생성 EI-MS 스펙트럼에는많은피이크가존재 조작이간편하고재현성이좋기때문에일반적으로널리사용. 분자의안정성이낮은경우 M + 가나타나지않기도하고또는극히약해서확인할수없는경우도있음. 물질이비교적휘발성이어야함. 고분자물질, 단백질할수없음

8 B. Chemical Ionization (CI) EI보다비교적온화한이온화방법 시약기체 (reagent gas) 라고하는특정기체를 0.1~1 torr 정도의압력으로이온화실에도입하고이를전자빔으로에너지를가하면시약기체로부터많은이온들이먼저생성되고이이온들이시료분자와충돌하여이온-분자반응을일으켜시료분자를 (M+H) + 로이온화시킴. 물질이비교적휘발성이어야함.

9 C. Desorption Ionization (SIMS, FAB, & MALDI 가속함 ) 사용 Beam 종류 : SIMS: Cs + or Ar +, FAB: Ar or Xe, MALDI: UV or nitrogen laser 비휘발성고분자이온화법 SIMS 와 FAB 에서확인되는분자이온형태 : (M+H) +, (M-H) -, (M+Na) +, (M+K) +

10 D. Electrospray ionization (ESI) 비휘발성, 고분자, 열에불안정, 극성화합물이온화시키기에 DI 보다더유용하게사용됨 사이의저분자화합물이온화도가능 Thermospray ionizarion (TSI) 과유사 전기전도성액체에전장을가하면액체가대전하여미세한液滴으로분무되는현상이용. 거의상압이므로 LC-MS에적용하기적합

11 4. Mass Analyzer mass analyzer의종류 A. magnetic sector mass analyzer B. Double-Focusing mass analyzer C. Quadrupole mass analyzer D. Time-of Flight mass analyzer MS 이름 : ionization method 와 mass analyzer 에따라정해짐 ex) MALDI-TOF-MS, ESI-Quad-MS,

12 A. magnetic sector mass analyzer 가속화된운동에너지 m: ion 의질량, n : 이온의속도, e: 이온의전하, V: 이온 - 가속판들사이의전위차 Magnetic field 에서 charged particle 은 curved flight path 로설명될수있다 H: strength of magnetic field r: radius of curvature of the path m/z

13 C. Quadrupole (mass filter) compact, less expensive, low scan time (<100 ms) - by 5-15V acceleration-pass stability of ions, mass limit 2,000

14 D. Time of flight (TOF) v=(2vz/m) 1/2

15 5. Mass Spectrometry Ion detector 종류 질량분석기를통과한이온에대해전기적신호로받을수있는부분을검출기라함 1) Photographic Plate 2) Faraday Cup detector 3) Electron Multiplier 4) Photomultiplier Detector 5) High Energy Dynode Detector 6) Array Detector 7) Microchannel Plate (MCP) GC-MS: GC 가 Mass Spec 에연결된것으로 Mass Spec 이 detector 의역할. LC-MS: HPLC 가 Mass Spec 에연결된것으로 Mass Spec 이 detector 의역할.

16 6. Determination of molecular weight ion mass 값을알아야한다. (exact mass (molecular weight = 단순 chemical atomic weight 의합 )) Mass spec 은 most common isotope 을함유하는경우와 heavy isotope 을함유하는 경우구별가능 fragment ion 과 M+ 와구별. molecular ion peak (M+) 확인하는요령 1. isotopic peak 제외하고가장 highest mass 가 M+ 2. isotopic peak 는주로 M+ peak 보다 lower intensity 3. Nitrogen Rule: N 을짝수 (no nitrogen 포함 ) 함유하는물질의 Mass = 짝수 mass value N 을홀수함유하는물질의 Mass = 홀수 mass value 4. Br 또는 Cl 함유물질일경우 : isotopic peak 가특징적 molecular weight: 60 C 3 H 8 O C 2 H 8 N C 2 H 4 O CH 4 N 2 O OH 함유물질은 dehydration 이잘일어남 à highest ion peak + 18 Acetate 는 acetic acid (60) 를쉽게잃음 à highest ion peak + 60 항상그런것은아님 물질마다특이한 fragmentation pattern 을참조해야함

17 A. Precise Mass Determination To determine intensities of M+1, M+2 peaks N-methyl-p-nitroaniline 예제 ) - O N + O NH C 7 H 8 N 2 O 2 Exact Mass: Mol. Wt.: m/e: (100.0%), (8.7%) C, 55.26; H, 5.30; N, 18.41; O, M+1 peak intensity = # C x natural abundance(%) + # H x natural abundance(%) + # N x natural abundance(%) + # O x natural abundance(%) = 7 x x x x 0.04 = 8.5 % of M+ peak

18 * Table: natural abundances of common elements and their isotopes Element Relative abundance Hydrogen 1 H H Carbon 12 C C 1.08 Nitrogen 14 N N 0.38 Oxygen 16 O O O 0.20 Fluorine 19 F 100 Silicon 28 Si Si Si 3.35 Phosphorus 31 P 100 Sulfur 32 S S S 4.40 Chlorine 35 Cl Cl 32.5 Bromine 79 Br Br 98.0 Iodine 127 I 100

19 B. Relative abundance of Cl on molecular ion 35 Cl: 100% 37 Cl: 32.5% (M) (M+2) For compound with one Cl: M/(M+2) 3/1 For compound with two Cl: M: 100%, M+2: 65%, M+4: 10.6%

20 C. Relative abundance of Br on molecular ion 79 Br: 100% 81 Br: 98.0% (M) (M+2) For compound with one Br: M/(M+2) 1/1 For compound with two Br: M: 100%, M+2: 195%, M+4: 95.4%

21 Table: relative intensities of isotope peaks for various combinations of bromine and chlorine Relative intensities Halogen M M+2 M+4 M+6 Br Br Br Cl Cl Cl BrCl Br 2 Cl Cl 2 Br

22 Fragmentation Patterns at Functional Groups* - to determine Molecular Formulas A Alcohols * Phenol (MW=94) 일경우 : Molecular ion peak (M+): strong Fragment ion: M-1, M-28 (= M-CO), M-29 (= M-HCO ) M + M-28 M-29

23 B. Ethers Molecular ion peak (M+): weak Fragment ion: a-cleavage, M-31, M-45, M-59, etc m/z=43, 59, 73, so on. H 3 C CH 3 C H a CH 3 O C H CH 3 MW=102 M-15 C 3 H 7 + CH 3 HC O + CH 3 C H CH 3 CH 3 HC OH + (45 m/z) + HC CH 2 CH 3 M-15 M +

24 C. Aldehydes for aliphatic Molecular ion peak (M+): weak Fragment ion: M-29 (= M-HCO ), butyraldehyde O M-43 (M-CH 2 =CH-O ) m/z=29, 44 M-HCO H 2 CH 3 C CH 2 H-C O + + CH C H 3 CH 2 CH 2 a-cleavage 72 m/z 29 m/z b-cleavage CH 3 CH CH 2 =CH-O McLafferty rearrangement R R CH CH H C H2 O H R R C C H H + CH 2 =CH-OH 44 m/z M-29 M +

25 C. Aldehydes for aromatic Molecular ion peak (M+): strong Fragment ion: for aliphatic: M-1, M-29 (C 6 H 6+ )

26 D. Ketones Molecular ion peak (M+): strong Fragment ion: for aliphatic: M-15, M-29 (=M-ethyl), M-43, m/z=43, 58, 72, 86, etc for aromatic: m/z=105, 120 M-29 M +

27 D. Ketones Molecular ion peak (M+): strong Fragment ion: for aliphatic: M-15, M-29 (= M-HCO ), M-43, m/z=43, 58, 72, 86, etc for aromatic: m/z=105, 120 acetophenone Additional a-cleavage C 6 H 5 CO + : m/z=105

28 E. Carboxylic acid Molecular ion peak (M+): weak for aliphatic, strong for aromatic Fragment ion: for aliphatic: M-17 (=OH loss), M-45 (=COOH+ loss) m/z=45, 60 (McLafferty rearrangement) for aromatic: M-17, M-45, M-18 R R CH CH H C H2 O OH R R C C H H + CH 2 =C-OH OH Butyric acid MW=88 60 m/z

29 E. Carboxylic acid Molecular ion peak (M+): weak for aliphatic, strong for aromatic Fragment ion: for aliphatic: M-17 (=OH loss), M-45 (=COOH+ loss) m/z=45, 60 (McLafferty rearrangement) for aromatic: M-17, M-45, M-18

30 f. Alkyl halides * Strong M+2 Cl: M: M+2 =3:1 Br: M: M+2 =1:1 Fragment ion: loss of Cl or Br, loss of HCl, a-cleavage

31 f. Alkyl halides * Strong M+2 Cl: M: M+2 =3:1 Br: M: M+2 =1:1 Fragment ion: loss of Cl or Br, loss of HCl, a-cleavage

32 7. Liquid Chromatography-Mass Spectrometry (LC-MS) 의원리 GC/MS와는달리 LC-MS 시스템은훨씬후에실용화 ß LC는액상, MS는기상에서또는 LC는상온, MS는섭씨 100~350도에서조작되도록고안 LC-MS 종류 - Thermospray (TS) LC-MS - Electrospray Ionization (ESI) LC-MS - Atmospheric Pressure Ionization (API) LC-MS - CFFAB(Continuous Flow Fast Atom Bombardment) LC-MS 등이개발됨

33 8. Liquid Chromatography-Mass Spectrometry (LC-MS) 의응용 LC-MS는신속성, 고감도 (high sensitivity), 선택성 (selectivity) 등의특성들로인해의약품개발의각단계에서중요한도구로사용 새로운선도후보물질 (lead candidate) 을찾기위한 drug discovery 단계에서의약품분석의주된역할은 (1) target identification (2) lead identification (3) lead optimization이됨. LC-MS는합성선도화합물의신속한분자량확인에서부터정밀한구조분석에이르기까지다양한범위에서 proteomics, glycoprotein mapping, 천연물확인및스크리닝, combinatorial/medicianl chemistry support, in vitro/in vivo drug screening, 대사안정성시험등의여러분야에응용되어지고있음

34 8. Liquid Chromatography-Mass Spectrometry (LC-MS) 의응용예 (1) LC-MS/MS 를이용한 2-DE 에서 분리된단백질의확인. (A) 2-DE에서분리된단백질 spot의 capillary HPLC로부터얻어진 base peak profile (B) 두번째주요피크 (residue 60-70) 의 MS spectrum (C) m/z 피크의 LC-MS/MS product ion spectrum ( 출처 : Arnott et al., 1995).

35 9. Gas Chromatography-Mass Spectrometry (GC-MS) 의원리 GC-MS 에서는기체크로마토그래프로머무름시간에따라분리된성분에대하여각각의질량스펙트럼을측정 (GC 가검출기로서질량분석계가사용 ) 휘발성성분이공존하는혼합물의분리분석 질량분석기는 10-5 ~10-6 torr 정도의진공에서작동 à GC 와 MS 장비를연결시키는인터페이스 (interface) 기술이우선적으로개발될필요 분리기 (separator) 를사용하여대부분의운반기체를제거하고분리된성분만질량분석계에도입 à 젯트형분리기 (jet separator): 저분자유기물은운반기체와함께확산됨으로감도저하초래 GC 모세관컬럼 (capillary column) 사용 : 유속을적게함으로서 GC 와 MS 를직접적으로연결할수있으며, GC 컬럼의분리능변화없이 MS 에서검출할수있는장점 비휘발성 이란질량분석기이온원의온도와압력에서충분한증기압을갖지않거나, GC/MS 에의한분석에서 GC 컬럼을통과할만한충분한휘발성을갖고있지않는물질을의미 비휘발성물질을 GC-MS 로분석하기위해서유도체화반응 à 화합물의열적안정성제고가능, 크로마토그래픽분리능을향상가능, 높은감도을제공가능, 질량분석시특정한이온을발생시켜구조분석에중요한정보를제공가능

36 감사합니다 References: 1. Introduction to spectroscopy, 4th ed. Pavia et al, Thomson Learning (2008) 2. Pharmaceutical analysis, 2nd Edition, David Watson, Elsevier (2005) 3. Daniel C. Harris, "Exploring Chemical Analysis" Third ed., W. H. Freeman and Company, New York (2004) 4. 약품기기분석학 (Pharmaceutical Instrumental Analysis, 신일상사 (2007) 5. Young In Analytical Applications 6. Agilent 자료 /Shiseido 자료