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- 0 C 3 C) 0 I I 1 1 $ -. - r Q I Quattro microTM Course Outline Morning Afternoon Introduction: QM Overview, Instrument Tuning:

  1. _[xiEi _ _ _ _ Z TM SPRAY’ Source E l e c t r o s p r a y - Exhaust Sample Cleanable - : Baffle Isobh:n Nebuliser Desolvation Source Enclosure Rotary Turbomolecular Pump Pumps Droplet Formation in Positive Ion Electrospray More Negative Ions than Positive Ions + + + + -- +_+ - - - -+- ++ L i q u i d Ø - - ++ +- _++ + ±++_++ _+ +++ ++ __+ +__+__; +___ Positively + + Charged + + Droplets Taylor Cone More Positive Ions than Negative Ions High Voltage Electrospray Probe Tip 16 Quattro micro Training Course

  2. The electrospray droplets carry positive c h a r g e s away from the c a p i l l a r y tube. To balance this flow of p o s i t i v e charges, electrons flow out of the c a p i l l a r y tube. T h e s e electrons come from negative i o n s close t o the surface of the capillary wall via an electrochemical oxidation reaction. Electrospray can be thought of as an electrochemical process. Example of reaction that can occur at the c a p i l l a r y w a l l : 20H—H 2 0+0+2e E l e c t r o s p r a y Droplet Undergoing Fission Solvent Coulom bic”; Evaporation Fission I I’ Charge resides o n the surface of t h e d r o p l e t . Solvent evaporates from t h e droplet and t h e droplet shrinks until the c h a r g e density o n t h e surface reaches a point w h e r e the repulsive force b e t w e e n charges exceeds l i q t h e u i d surface tension that holds the drop together. At that point, t h e drop fissions and a set o f s m a l l droplets are expelled f r o m t h e m d r o p a i n l e t . 17 Quattro micro Training Course

  3. l Droplet Undergoing Coulumbic Fission A c t u a .....•• Photo r o m Rough Sketch o f f Tang, Analytical Chemistry, 64, 9 7 2 A ( 1 9 9 3 ) P. Kebarle and L. droplet will estimated, that in t h e fissioning process a charged It is mass. of 15% f its charge but as little as 2 % o f its lose o n t h e order o to work best with solutions Electrospray tends percentage of organic s o l v e n t s that have a high acetonitrile or methanol, though the s u c h as solution cannot be totally organic. The solution must have some aqueous content. Solutions must have some ions i n it for to work. electrospray e o u s Fortunately most solutions that h a v e an a q u species s u c h component will h a v e some i o n i c a s hydronium/hydroxyl ions and sodium ions. 18 Quattro micro Training Course

  4. Models f o r formation o f Gas Phase Ions from Droplets Ion Evaporation Model Through evaporation and f i s s i o n i n g , droplets reduce in size t o 10-20 nM in Diameter Ions then ‘evaporate’ from the d r o p l e t ’ s surface. The more ‘Surface Active’ a molecule i s , the more readily i t will f o r m ions i n electrosp ray. Charged R e s i d u e Model Droplets continue t o lose solvent molecules through evaporation till a charged residue remains. For an analyte of the form M X , the charged residue will be of the form: (M) n(M)Qm Electrospray Ions Positive Electrospray Ions are produced by the a d d i t i o n t o a molecule of p o a s i t i v e l y ion (e.g H+, N H 4 + , Na+). These p o s i t i v e l y charged ions t h a t are added are often referred to as ‘adducts’. H H C 3 H L i d o c a i n e 3 C H Negative Electrospray Ions are most often produced by the removal of a proton (hydrogen ion) from a molecule. C 3 H C 3 H I b u p r o f e n +H+ 3 C%(’r H H 19 Quattro micro Training Course

  5. Electrospray and Ions in Solution Electrospray is a solution process. Molecules that have a greater tendency to ionize in solution will tend to have stronger electrospray signals. This is why certain additives to mobile phases in LC/MS analyses can enhance electrospray signals. An example of this is addition of an acid (e.g. formic acid) to the mobile phase in positive electrospray LC/MS analyses. This can often result in a stronger electrospray signal by aiding in the protonation of analytes in solution. 13-Cyclodextrin - Electrospray Example HO OH HO0H HO OH HO\f / / 13-Cyclodextrin is a ring of 7 OH OH HO 40 HOO HOH Oxygen Linkages OH OH are Numbered 20 Quattro micro Training Course

  6. Positive Ion E l e c t r o s p r a o f 13-Cyclodextrin y Quattro microTM BetaCyDex_5 1 (10.018) 2: Scan ES+ 38 ( M + N H 4 ) + 1152 6.92e6 100 1135.37 (M+H)+ e Voltage=45 V %. 0 BetaCyDex_5 1 (9.983) 1: Scan ES+ 1157.36 6.36e6 100 (M+Na)+ (M+K)+ ii7j3.36../ 1158.37 I % / Cone Voltage=140 V iL r. -.—.——i miz 1120 1130 1140 1150 1160 1170 1180 10 pg/mL 6-Cyclodextrin in 20/80 Acn/20 mM NH4 Acetate pH=4 in Water infused at 10 pLjmin Negative and Positive Ion Electrospray of 6-Cyclodextrin Quattro microTM BetaCyDex_6 1 (2.186) Scan ES- 1133.36 8.73e6 (M-H)- Negative Ion Ele&ospray p34.37 Cone Voltage = 100 V • I I BetaCyDex5 1 (10.018) 2: Scan ES+ 113537 5.53e6 100 (M+H)+ Positive Ion Electrospray 36 44 Cone Voltage =45V %. 0 i m/z I 1125 1130 1135 1140 1145 1150 10 pg/mL B-Cyclodextrin in 20/80 Acn/20 mM NH4 Acetate pH=4 in Water infused at 10 pL/min 21 Quattro micro Training Course

  7. Samples A n a l y in ES z e d m o d e Typical ES Positive Ion Samples Peptides and proteins Small polar molecules Drugs and their metabolites Environmental contaminants Dye compounds Some organometallics Small saccharides Typical ES Negative Ion Samples Some proteins Some drug metabolites (e.g. conjugates) Oligonucleotides Some saccharides and polysaccharides A t m o s p h e r i c Pressure C h e m i c a l Ionization (APcI) Low molecular weight (<1000 DaJ Singly charged species Fragmentation, even at low cone voltages Mobile phase can be non-polar (normal-phase chromatography) 22 Quattro micro Training Course

  8. __ ____________________ APcI Source Cowori DiSchar9o Pin f I CCnanbTc Ezhsut —* liner 41 NbulI5es Ocolyation ir ii []1 Restrictoi }tA4____ 1 II I Enclosure —. Rolury Turbomelocuku Pump Pumps APCI Probe Design Fused Silica Capillary Plasma of Stainless Tube (Sample flows through) Ionized N 2 / and °2 Ions / I 1, I - $ 4 - / Corona Pin Drying APcI Sheath Nebulizing (Voltage Gas Gas Gas Applied) 23 Quattro micro Training Course

  9. ionization A P C I Higher • temperature, more aggressive ionization. • Solvent molecules are i n the g a s p h a s e . • Ionization takes p in l a c e the plasma. • Goal o f the nitrogen is to evaporate solvent expelled from fused silica. • M a y be more sensitive than electrospray with some non-polar m o l e c u l e s A P c I I o n s In positive ion APcI, ions similar to those formed in positive ion electrospray are formed. For example: (M+H)+ or (M+Na)+ In negative ion APcI, the CM-H)- ion formed in negative ion electrospray i s also produced. Also in negative ion APcI, free electrons are formed. Certain types of molecules can pick u p one of the free electrons produced by the corona pin and become negatively charged without a change in mass. This process is sometimes referred to as “M+.” or “M plus dot”. I I 2 4 Quattro micro Training Course I

  10. APCI v e r s u s Electrospray 100,000 -c 3 ) 1000 a) 0 Non Polar P o l a r APCI v e r s u s E l e c t r o s p r a y 100,000 C) a) 1000 U ) 0 0 Non Polar P o l a r Quattro micro Training C o 25 u r s e

  11. A P c I versus Electrospray 100,000 -I c3) a) 1000 a) 0 0 Non Polar Polar APCI versus Electrospray APcI Electrospray Ionization Gas Phase Process Solution Phase Process Probe Fused Silica Capillary Stainless Steel Capillary Potential Applied to Corona Pin Applied to Capillary Process Probe heater vaporizes Spray of charged droplets the liquid. produced. All molecules are Liquid is evaporated from now in the gas phase. the droplets. Corona pin produces Then droplets split into nitrogen ions. smaller droplets. Molecules are ionized When the droplets get when they collide small enough, ions enter with the nitrogen ions. the gas phase. 26 Quattro micro Training Course

  12. API versus Electrospray ( c o n t i n u e d ) APcI Electrospray Fragments More vigorous ionization. ‘Gentler’ ionization. More fragments produced. Less fragments produced. Sample Types Low MW<1000 Small & Large Molecules Can be less polar. Tend to be more polar. Charges Usually Singly Charged. May be Multiplied Charged. Flow Rates 0.2 - 2 mL/min 0.001 - 1 mLJmin Temperatures Source “.‘ 120-140 °C Infusion:Source “-i 80 °C Probe 450-550 °C Desolvation 120°C “ HPLC: Source 120 °C “ Desolvation 350 °C ‘ P o s i t i v e o r Negative? Compound Type Easiest Formed Ions Basic C o m p o u n d s (-NH 2 ) (M+H) P o s Ions Acidic C o m p o u n d s H 2 ( - C O , -OH) (M-H)- Neg Ions 27 Quattro micro Training Course

  13. ______ _________ ______ _ _ _ Waters Z-Spray Source I I I MICROMASS MS TEC.HIOL.CC US I I RF Transfer Optics Desolvation Cleanable Sampling Hexapole Ion Bridge Vent Baffle Gas Manifold Cone •\•--* iO mBar I • I I Isolation Valve r : z z . I Restnctor I yfll Z SPRA I I Ion Block 1 mBar I 28 Quattro micro Training Course i

  14. R F Transfer Optics Cleanable Desolvation Sampling Vent Hexapole Ion Bridge B a f f l e Gas M a n i f o l d Cone 1O mBar I E E \ ‘-*- 4 _ Isolation - V a l v e / ZSPRAY Extraction I o n Block 1 mBar Cone “ . ‘ Neutral solvent evaporating iconS-shaped reçon from i n i t i a l s p r a y ) 4 I Skimmer Why Solution inlet t o m - analyzer electrospray source —-—fr b of m a s s - Z-S p r a y ? b\ spectrometer — Ion b e a m Neutral solvent evaporating ( c o n e - s h a r e g i o n p e d from iniaL s p r a y ) L Sokiuiri inlet to electrospray source —b- - - S k i m m e r o h t i c e o f m a s s s p e c r r o m e t e r Z - s h a p e d i o n beam t More ions make it into the Z-Spray source 29 Quattro micro Training Course

  15. Source - Electrospray Z SPRAY TM Sample Rotary Turbomolecular Pump Pumps Probe Position Sample Sample Cone \... Capillary 0.5mm 4mm 8mm Probe Tip 30 Quattro micro Training Course

  16. Electrospray Probe Tip Stainless Steel Tube \/ Stainless Steel Capillary Liquid I Electrospray ‘Plume’ Electrospray Probe Tip Nebulizer Gas I Liquid ‘ I Electrospray ‘Plume’ Nebulizer Gas 31 Quattro micro Training Course

  17. E l e c t r o s p r a y P r o b e T i p D e s o l v a t i o n G a s N e b u l i z e r G a s L i q u i d . 2 _ _ I Electrospray N e b u l i z e r G a s ‘Plume’ D e s o l v a t i o n G a s Desolvation Gas Flow N i t r o g e n I H e a t e r Heater W i N i t r o g e n 32 Quattro micro T r a i n i n g Course

  18. _ _ Quattro Ultima a n d Quattro LC: Sample C o n e a n d Cone G a s N o z z l e — ESP or . b APCI Probe Plume of t . , . ‘ : Ions, • I . & • Clusters, •L • and Stuff . •• • •.I: Ions with Fewer Clusters C o n e G a s which yields better SIN. Less Stuff Collects ‘P on the Orifice Cone 33 Quattro micro Training Course

  19. _ _ _ _ __________ ____________ _ _ _ _ _ _ _ I I I Cone G a s Example D e x t r o m e t h o r p h a n - I nqlmL DextroMTP 20 uL X T e r r a C8 1 Dextro 104 Sb (2,1.00); Sm ( M n , 2x1) MRM of Channels 2 55. o65 / ] 4.09 100 3 0 9 T I C S 53e3 260 2 6 0 00, Cone G a s = 125 I D e x t r o l o 5 5 b (2.1.00); Sm ( M n , 2x1) MRM of 2 Channels E S . 4.09 100 T I C 8.28e3 09’ % Cone Gas 175 I K 0. Dextro,1_065b (2,1.00); Sm ( M n , 2 s f ) MRM of 2 Channels E S . o60 4 0 9 T I C 8.43e3 C o n e Gas 225 = OextrQ_1_07 Sb (2,1 00); Sm ( M n , 251) MRM sf2 Channels ES. 100 4.09 TIC 7.28e3 Cone Gas fl% 275 Time 2 . 0 0 2.50 3 00 3 . 5 0 4 . 0 0 4 . 5 0 5 . 0 0 50 ‘ 5 ‘ Baseline N o i s e Magnified by F a a c t o r of 6 0 1 APcI Source Dtsharge L r — - — Sample C l e a n a b l e E x h a u s t -+ = B a f f l e Liner B S a m p A h l e r —‘ Isolabon Nbullser DesIvetion Gas Gas EnEL Rotary 1 u r b o m o 4 c c u t a Pump P u m p s 34 Quattro m i c r o Training C o u r s e

  20. in: 4 u N: 4 i1 ‘“ 5 S: 1 zz.i r * I.n”.,l,s’ Si.. . i. ZSPRAY Th ’ çLz 441 .4. Waters Mass Spectra and MS Data Acquisition Modes ‘, MICROMASS’ MS I EcHwoI.OG:r’; 35 Quattro micro Training Course

  21. Mass S p e c t r o m e t e r Data Acquisition M o d e s M S Modes MSIMS Modes • MSI Scan • Daughter Ion Scan Parent Ion Scan a . •MRM • Constant Neutral Loss(Gain) I I MS1 Scan / M S 1 Collision M S 2 / C e l l (No Argon) Scanning RF RF(-i-DC) 10 -100V m 1 m 2 m 3 3 6 Quattro m i c r o Training Course

  22. MS Spectra of an Infused Spectra of ‘fens Scan ES- MIX4INF2 1 (1019) 598e6 205.1 100 Ibuprofen (M-H) 253.1 243.1 Flurbiprofen Ketoprofen (M-H) (M-H) C 245 255 260 265 270 275 280 180 185 190 195 200 205 210 215 220 225 230 235 240 250 Monoisotopic versus Average Molecular Weight Monoisotopic Molecular Weight is calculated using the atomic weight of the most abundant isotope of each element. Average Molecular Weight is calculated using the average atomic weight of each element taking into account the relative abundance of its isotopes. For example: Naturally occurring carbon is 98.93% Carbon-12 (AtWt=12.0000) and 1.07% Carbon-13 (AtWt=13.0034). In calculating a monoisotopic molecular weight, each carbon would add 12.0000 to the molecular weight. The average atomic weight of carbon is: (0.9893)(12.0000) + (0.0107)(1 3.0034) = 12.0107 So in calculating an average molecular weight, each carbon would add 12.0107 to the molecular weight. The molecular weight listed on reagent bottles is an average molecular weight. Quattro micro Training Course

  23. Mass Spectra Different I s o t o p i c Forms of an Analyte - MIX4_INF3 1 (2021) Sm (SG, 2x0 50) Scan ES- Thia_Scan_Ol 79 (3384) Sm (SG, 2x0.50); Cm (77:84-(3 243.0 3.33e6 292 464e5 100 100 Thiamethoxam (M+H) uthiprofen 0 i m,z a miz 240 241 242 243 244 245 246 247 248 290 292 294 296 C H 13 0 2 F 15 5 SC1 3 0 N 10 H 8 C —9L eycWa i l . c t ç * V U a M a s s Spectrometer A c q u i r e s Spectra in the ‘Profile’ or ‘Continuum’ Mode I j 1 J J L ‘Continuum’ Data DOD Ca / : \ a \/\ D0 / Do 0 mri 608 609 610 611 608 609 610 611 E x a m p l e of Profile D a t a a c q u i r e d If directly stored by MassLynx, at 1 6 p o i n t s D a p e r l t o n t h i s i s ‘Continuum’ Data Example: S u p p o s e you a r e acquiring s p f r o e c t r a m 300 t o 700 a m u a n d t h a t you a r e t a k i n g o n e c o m p l e t e s p e c t r u m e v e r y s e 1 c . T h e n 60 separate c o n t i n u u m s p e c t r a w i t h r a n a g e of 400 a m u w i l l be s t o r e d e a c h m i n u t e of a c q u i r e d d a t a . 3 8 Quattro m i c r o T r a i n i n g Course

  24. Converting Continuum Data to Centroid Data The mass spec can also take each profile or continuum spectrum it has acquired and convert it ‘on the fly’ to centroid data. In this process, the center of each spectral peak is determined and only information on the center of each peak is transmitted to MassLynx. ‘Centroid’ Data . n 0 608 609 610 611 608 609 610 611 un 1:x cac 1 •—: Data Acquisition Modes - ce-L k • Continuum - also known as “Profile” data series of spectra are acquired are stored individually largest data file size shows peak shape can handle signals that vary with time (e.g. LC Peaks) • Centroid - also known as “Stick” data sees profile data but instantly converts it to stick data, smaller data file size than continuum data gives no peak shape information. • MCA - Multi-channel Acquisition is continuum data summed into one scan smaller data file size than continuum but preserves the peak profile. assumes there is a constant signal (infused sample) 39 Quattro micro Training Course

  25. __________________________________ _______________________ _________________ _______________________ ___________________ ________ I I I Which Data Acquisition Mode? I Maximum Scan Speed Mode Typical Use I Atl6ptsIDa GC, OpenLynx, Centroid 1000 amulsec FractionLynx, high I concentration samples 1000 amulsec Multiply charged (EPCAS) I species, non-time MCA resolved data 400 amulsec (e.g. Infusion) (TDAT) I 1000 amulsec Multiply charged (EPCAS) Continuum species, time resolved data 500 amulsec I (e.g. HPLC) (TDAT) I I Mass Spectra of LC Peaks I 000 nqimL Thiamethoo4m end Metabolite hia_Scan_Ol 79 (3.384) Sm (SG. 2x0.50): Cm )75:85-(437l+1 101 Scan so. na_Scan_SI Son (Mn, 2x1) 2 2 3.76cr 1 377 i’ 2.860 Spectrum of Peak Thoarnethonano 340 with RT of 3.40 (MCH) I From CVF all NCMdaS r 2 I mono G-’ L 20 300 350 400 480 500 mhia_Scan_31 101 (3.759 Sm LSG. 2x0.50): Cm(97’104-(437fa1I 20 584e7 100’] Full Scan Spectra of LC Analysis of a Mbnlee VF Cone Voltage (MOO) Standard Solution of Thiamethoxam FragrnontntOfl I and one of its Metabolites. I Spectrum of Peak w4h prof 377 Spectra taken using I second scans Fr0wCVF from 100 to 400 Da. I AI.m.r.. 19 of, 160 180 200 220 240 200 280 300 320 340 Spectra of two LC Peaks. I I 40 Quattro micro Training Course I

  26. - Infused Sample vs LC MS Sample MS Scan Thiamethoxam Scan ES+ THIA_INFUS_02 1(0.176) Sm (SG, 2x0.70) 143 5.85e6 181 10 M+Na 197 Spectrum from M+H 314 205215 250 292 Infusion 330 229 241 .jap• 391 Thia 1F29 011 79(3.384) Sm (SG. 2x0,70): Cm (76:84) Scan ES+ 2 2 4.1 7e5 100 M+H Combined Spectra CVF from Peak at 3.4 mm % M +Na 211 in LC/MS Analysis 186 197 156 246 314 Scan ES+ Thia_i F29_0i 1 79 (3.384) Sm (SG, 2x0.7d); Cm (76:83(45:73+8€ 96)) 2 ‘2 4.55e5 100 Combined Spectra from M+H Peak at 3.4 mm in CVF LC/MS Analysis with 211 M+Na Spectra Subtraction 246 314 IL. m/z 300 320 340 360 380 140 160 180 200 220 240 260 280 Cone Voltage Fragmentation Fragments from Collisions along with ‘Unfragmented’ Ions Produced Ions by ESP or APCI Ions which are accelerated Cone Approx by the cone voltage, collide with Nitrogen 20-1 OOV molecules 41 Quattro micro Training Course

  27. I Example of Cone Erythromycin : MW 7 3 3 D a = Voltage Fragmentation micro Q u a t t r o ERYTHRO_010_MS 1(1.530) Scan ES+ 7 4 1.34e8 100- C o n e V o l t a g e = 3 0 V U %- 140 6 159 193 5 7 718 576 0— I I ERYTHRO_011_MS 1(1.530) Scan ES+ 158 2.20e8 ioo 1 C o n V e o l t a g e 5 0 V = 576 I % L 5 5 8 y6 1 19 ERYTHRO_012_MS 1(1.530) Scan ES+ 158 6.62e8 1001 7 5 C V o l o n e t a g e V = % 1 1 6 19 0— ‘.‘—‘.‘.i— irrI/z 150 200 250 300 350 400 450 500 550 600 650 700 750 800 0 Me .Me Me\,Me OH Me\,Me 0 Me Z7Me H Z 7 M e - o OMe 0— - 157Da Me - - M e 7 3 3 D a Me Cone Voltage o Fragmentation MeL.Me LyH Me\NMe In M e i E r y t h r o m y c i n O 0 1 1 M e OO Me 575Da H 42 Quattro m i c r o Training Course I

  28. R e c o g n i z i n g Multiply Charged I o n s Mass spectrometers operate o n the basis o f mass-to-charge r a t i o (mlz). Mass assignments are n o r m a l l y made assuming a single charge per i o n ( e . g z so . 1 mlz m) = = Single charge mlz (M+H) = Double charge mlz ( M + 2 H ) / 2 = n charge ( M m l z + n H ) I n = Modified 6-Cyclodextrin Multiplied Charged Example - Modified 1 3 - C y c l o d e x t r i n . HO OH 0 Added functional groups a r e HO shown i n t h e dashed OH circles. o Sample from 0 N 2 H OH HO Prof. P a i n Smith, Baltimore County H O \ f OH 8-Cyclodextrin HOZN.,o H)o:o0H a r e Numbered OH Quattro micro Training Course

  29. I I I Modified 6-Cyclodextrin - Multiplied Charged Example I Quattro microTM SH_263_MS_202 1 (2.091) Scan ES+ 1217.5 1.41e7 Positive Ion Electrospray (M+H)+ Cone Voltage = 100 V 1l8.5 %• 546.1 690.2 ‘ U. Scan ES+ SH263_MS201 1 (2.091) 609.2 ba 4.29e8 (M+2H)++ Q97 Positive Ion Electrospray Cone Voltage = 55 V • %. o—,—————,— 600 700 800 900 1000 1100 1200 1300 10 pg/mL Modified 6-Cyclodextrin in 20/80 Methanol/0.1% Formic Acid in Water infused at 10 pL/min Modified 13-Cyclodextrin - Multiplied Charged Example Assume Monoisotopic Mass of Analyte is M. Mass of Isotopic Forms (mostly from Cl 3’s) = M, M+1, M+2,,, 121748 100 Top Spectra For Singly Charged Ions (assume addition of H+) m/z of Isotopic Forms= M+1, M-I-2, M+3,,, 1219.50 1220.44 & (Monoiso MW=1216.45) i miz I I 1216 1218 1220 1222 60926 Bottom Spectra 100 For doubly charged ions, (assume addition of 2H+) °k mlz of Isotopic Forms = (M+2)12=M12+ 1, 10.27 (M+3)12=M12÷ 1.5, (M+4)12=M12+2, m/z 0 i i I I I 607 610 608 609 611 612 613 614 Quattro micro Training Course

  30. Multiply Charged Ions Substance P : Arg - Pro - Lys - Pro - Gin - Gin - Phe - Phe - Gly - Leu - Met - NH2 Substancc P SIJL7PEI I (Di1k S:M,, 2D.E: EL (I.EfEE) Scan ES. M+II1 4525 13417 $DE 117<1 ‘750 Example of Multiply Charged Ion 100 Electrospray Spectra of Horse Myoglobin mlz and Charge States 20 649 Shown 15 1131 21 808 14 1212 22 771 13 1305 12 23 1414 11 738 1542 9 24 1696 707 1885 c mlz ..— .-.--. — — —. — 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 Quattro micro Training Course

  31. M S 1 Scan (Review) M S 1 Collision M S 2 C e l l (No Argon) Scanning R f Rf(+ D C ) 10 - 1 0 0 V m 1 m 2 3 m M S 1 S c a n s a r e u s e d t o o b t a i n M a s s S p e c t r a SIR (Selected Ion Recording) M S 1 Collision M S 2 C e l l (No Argon) Fixed RF R f D C ) (+ 10 - 1 0 0 V m 1 m 2 m 3 Ss a r e u s e d t o m o n i t o r s e l e c t e d analyte(s) 46 Quattro micro T r a i n i n g Course

  32. SIR Example 10 nqlmL Thiamathoxam and Metabolita Quattro micro’ 0 Thia_1G07_007 Sb (21.00); Sm (Mn, 2x3) SIR of 2 Channels ES+ Thiamethoxam 100 291.8 1.07e4 MW=291 SIRof(M+H) mlz = 292 % Th’_1G07_007 Sb (2,1.00); Sm (Mn. 2x3) SIR of 2 Channels ES÷ 249.8 100 1.61 e4 Metabolite MW= 250 . .50 8.00 8.50 9.00 9.50 io.&5 Daughter Ion Scan MSI. Collision MS2 Cell (w/Argon) Fixed Scanning 5-40 eV m 2 m 1 I Determines Collision Induced Dissociation (CID) produced daughter ions of a particular parent ion 47 Quattro micro Training Course

  33. Daughter Ion Scan — Effect of Changing Collision Energy MS/MS Spectra of Chlorpheniramine (MW=274) Daughter Ions of m/z=275 25 M+H..., Collision Energy = 5 eV 100• 275 100 230 Collision Energy = 12 eV ° 275 0 100 230 Collision Energy = 17 eV °“° 0 150 160 170 180 190 200 210 220 230 260 240 250 270 280 290 Daughter Ion Scan — Effect of Changing Collision Energy (Cont.) MS/MS Spectra of 230 Clorpheniramine Collision Energy = 17 eV Ia Daughter Ions of m/z=275 M+H 0 100 Collision Energy = 30 eV 167 230 0 221_2O2 0 100 Collision Energy = 38 eV 167 180 201 230 194 0 ,,•i,Jz Il—I 150 160 170 180 190 200 210 220 230 260 240 250 270 280 290 48 Quattro micro Training Course

  34. Daughter Ion Scans — Propranolol Example MS/MS Spectra of Propranolol (MW=259) Daughter Ions of m/z=260 Daughters of 260ES+ X_PROPRANOLOL_MSMS 1(1.015) 260 1.63e7 100 116 183 M+H 7274 98 157 86 155 218 A AA 0 L I I I I I I I I I 260 280 60 80 100 120 140 160 180 200 220 240 MS/MS spectra can often be very complicated. Maximum daughter ion signal obtained using a collision energy of 18 eV. Increasing the collision energy beyond this level, led to more CID of the parent M+H ion, but also led to more CID of the daughter ions resulting is lower daughter ion signals. — Thiamethoxam Daughter Ion Scans From MS Scan of Infused Sample Scan ES+ THIAJNFUSO2 1(0.176) 5.57e6 1 1 197 100 Thiamethoxam i.7LLLLLJL 2 THIAJNFUS_03 1(1.017) Daughters of 292ES+ 211 3.90e6 lao Daughter Ion Scan % Daughter Ions of m/z=292 Thiamethoxam Ion 11 ii o I 1 Daughters of 250ES THIA_INFUS_04 1 (1.475) 6.9706 1 9 oo Daughter Ion Scan 1% Daughter Ions of m/z=250 Metabolite Ion mlz 290 300 310 320 330 150 160 170 180 190 200 210 220 230 240 250 260 270 280 Quattro micro Training Course

  35. I I I M S - M S o f Multiply Charged I o n s The most intense i o n s a r e normally used for MS-MS I even i f t h e y are multiply charged I Multiply charged i o n s may require higher collision gas pressures than singly charged ions I Fragment ions can be larger in apparent mass t h a n the multiply charged precursor i o n s I I V a n c o m y c i n H O O ( M + 2 H ) M)ç5 H 3 C 724.9 OH 0 0 0 O O I OH / p6,9 (M+H)+ OH HO Magnified 1451.6 I 14 by 15X 1452.6 17.9 1453.6 1418.5 h t - r i g - j C 4bI4.nh,z 500 700 800 900 1000 1300 1100 1200 1400 1500 5 0 Quattro micro Training Course I

  36. MS Spectra of Vancomycin VANCO_14_MS 1 (0.284) Sm (SO, 2x0.60) Scan ES+ 14506 6.39e5 100 0: 1453.6 mlz 1446 1447 1448 1449 1450 1451 1452 1453 1454 1455 1456 VANCO_15_MS 1 (2.055) Sm (SO, 2x0.60) Scan ES+ 7249 7259 2.34e7 100 % 727.4 7 .9 . m/z , . . 721 722 723 724 725 726 727 728 729 730 731 Vancomycin Daughter Ions of (M+2H) Ion (m/z= 725) Quattro microTM VANCO 04 MSMS 1 (2.055) Daughters of 725ES+ 144.1 x5 1.28e7 Spectrum Magnified by 5X Singly Charged from m/z = 600 to 1500 4— Daughter Ion 1307.3 %• Unfragmented 725.6 Singly Charged Doubly Charged Daughter Ion —b Parent Ion 1145.3 . 200 400 600 800 1000 1200 1400 51 Quattro micro Training Course

  37. ) Closer Look at Daughter Ions o f ( M + 2 H ) Ion (m/z=725) V A N C O 05_MSMS 1 (0.890) Sm ( S G , 2x0.60) V A N C O _ 0 5 _ M S M S 1(0.890) Sm ( S G , 2x0.60) 1442 5.59e6 1307.3 100 5.43e5 — 100 13053 nrO ‘., .— 130 I 1 3 0 9 . 1 i 0 iZ 0 i m/z 140 i 142 144 146 148 1304 1306 1 3 0 8 1310 Note: L M & HM of MS1 were opened up t o pass all isotopic forms of t h e m/z=725 i o n into t h e collision cell. ‘ I s o t o p e Peaks’ a r e 1 Da apart. C 3 H HO” P o s s i b l e O o Possible F r a g m e n t a t i o n c J L O F r a g m e n t a t i o n H -- —— —... CI I - / N NH INH CH 3 ‘ 0I CH 3 I NH 2 O H O b s e r v e d D a u g h t e r ions of / O H 7 m / z 725 OH P a r e n t ion a r e H O = s e e n at m / z 1 3 0 5 a n d 144 = 5 2 Quattro m i c r o Training Course I

  38. Example of MS/MS of a Doubly Charge Ion [Glul]-Fibrinopeptide B Glu-Giy-Vai-Asn-Asp-Asn-Glu-Glu-Gly-Phe-Phe-Ser-Aia-Arg GluFib 5 pmoiluL in AcnlWat infused: 4 uLlmin GluFib_Parent 1(2.191) Sm (SG, 2x0.50) Scan ES+ 785.7 x100 6,34e7 100 (M+2H)—4 MS Scan Magnified by 100 % 1570.4 + (M+H) —+ 333.1 480.1 1221.1 Ii I 1285.2 II I 684.1 I 0 i4Jmiz 200 400 600 800 1000 1200 1400 Example of Daughter Ions From a Doubly Charge Ion [GIul]-Fibrinopeptide B Giu-Giy-VaI-Asn-Asp-Asn-GIu-GIu-Giy-Phe-Phe-Ser-AIa-Arg 11 10 9 GiuFib 5 Dmoi!uL in AcnlWat infused: 4 uUmin GluFib_Parent 1(2.191) Sm (SG, 2x0.50) Scan ES+ 785.7 x100 6.34e7 100 j MS Scan (M+2H)’ Magnfied by 100 (M+H) -) 1570.4 33?1 480.1 1221.1 Iii I II 684.1 I 0 4 i.j:r#r..’.,,,,- •-,- 4 GIuFib_Dau 1 (3.370) Sm (SG, 2x0.50) Daughters of 786ES+ 333.0 289e6 ioo 186.9 Y’6684.0 y 7 8129 480.0 Daughter Scan of mlz=786 1% Yg Y 942.0 240.0 ‘11 ‘lO 1056.0 I 627.0 382.0 1171.0 1285.0 I I ill I m/z 200 400 600 800 1000 1200 1400 Quattro micro Training Course

  39. p cLL MRM (Multiple Reaction Monitoring) MS1 Collision MS2 Cell (w/Argon) Fixed Fixed 5-40 eV m, m MRM’s are used to monitor selected analyte(s) via their daughter ions m pie I Ion Chromatograms from SIR’s of m1z255 Mixiso I1314 022 SIR xl I Chxnncl ES. 1.31 TIC 100 5.95e6 / From a Sample that is / 60 nglmL Ketoprofen Fenbufen 60 nglmL Fenbufen % OOH Ketoprofen Fenbufen Ketoprofen 0 Th Time 0.80 0.90 1.00 1.10 1.20 1.30 1.40 1.50 1.60 1.70 SIR of I Chonnel EOn MIxIso_l SI 4_023 1.31 TIC 100 6.03e6 QOOH From a Sample that is / 60 nglmL Ketoprofen 6nglmLFenbufen / Both have a MW of 254 % Ketoprofen Fenbufen?? 0.00 0.óO 1.óO 1.10 1.20 1 30 1.40 1.50 1.60 1.70 54 Quattro micro Training Course

  40. _____________________ SIR - C o m p a r i n g MRM Example a n d I (cont.) hxIsh_1G14_023 SIR of 1 Ch>rmel ES. I,?\1 100 E0 From SIR of m1z255 , C Ti .. . . 080 0.90 100 110 120 130 1,40 150 160 1.70 Ion Chromatograms from hx.h> lGn4 24 MR&4 o2 Channol, 5:5’ 17 25525.25g2 SIR and MRM Analyses 100 of a Sample that is From MRM of 60 ng!mL Ketoprofen mIz 255> 209 j Ketoprofen 6 ng/mL Fenbufen .% SEnis L1G14...024 MRM 012 Channel, ESC 142 255,25>2372 100 0 0604 [\ / \ From MRM of Fenbufen rn!z=255>237 % ,>_,-nn;/” 580 0.90 100 110 125 130 140 150 170 1.60 hia 1007 007 Sb (21.00); Sm (Mn. 2x3) SIR of 2 Channels ES* C o m p a r i n g 100 291.6 1.076’ MRM and SIR Thiamethoxam Example 2 hiaiGO7_007 Sb (2,1.00); Sm iMn, 2.31 SIR 012 Channels ES> SIR’sofalonglmL Standard Solution of Thiamethoxam & Metabolite iomS 50 800 8,50 900 950 Ela 002 Sb 12 1.00 1. Sm (Mn, 2.31 SIR 012 Channels ES’ SIR’s of Sample of JNo] 10 nglmL Thiamethoxam - [ m e t h o x a J & Metabolite in a Fruit Matrix Eia_1007_002 Sb (2.1.00); Sm (Mn, 2>3) SIR 012 Channels ES. 100 249.0 \ 1 42e4 Peak Labeled an impurity in Metabolite??_[—-.-* the fruit matrix sample is also present in blank matrix samples. Time . 511 900 550 91111 550 1000 55 Quattro micro Training Course

  41. Comparing MRM and SIR Example 2 (Continued) MRM’s of Sample of 10 ng!mL Thiamethoxam & Metabolite in a Fruit Matrix Mi SpIted with Lww Stnd.,d OuattrD mIvro’ Thia_1G04313 Sb (21.00 ); Sm (Mn, 2x3) MRM of 2 Channels ES+ 291.8> 210.8 433 famethoxam Thi’ 1G04 313 Sb (2,1 .00 ); Sm (Mn, 2x3) MRM of 2 Channels ES÷ 100 249.8 > 168.8 463 oiitj-, % i0.d6w y.50 8.00 8.50 9.00 9.50 Peaks Labeled as Thiamethoxam and Metabolite are not present in blank matrix samples. Parent Ion Scan Consider a class of compounds that are similar in structure: Different Compounds Different Same That Are Somewhat Neutral Charged Similar In Structure Fragments Fragment CID -4 + CID -4 + Parent Ion Scans can be used to detect those compounds whose molecular ions produce the same charge fragment. 56 Quattro micro Training Course

  42. PAR (Parent Ion Scan) MS1 Collision MS2 Cell (w/Argon) Scanning Fixed 5-40 eV m, m 3 I m 2 Find ions that will produce via CID, daughter ions with a particular m/z Parent Ion Scan Example: Lets look at the MS/MS Daughter Ion Scan of a Beta Blocker, Propranolol X_PROPRANOLOL_MSMS 1 (1.015) Daughters of 260ES+ 260 1.63e7 100 Major Daughter Ion at m/z=116 M+H 98 157 86 155 141 J I 218 J 165 Au 1 rnlz I I i i i i 60 80 100 120 140 160 180 200 220 240 260 280 57 Quattro micro Training Course

  43. Example of B e t a B l o c k e r s whose M+H Ion All Produce a m / z = 116 Daughter Ion D a u g h t e r I o n of m / z = 116 p r o d u c e d CID b y at spot indicated by line. —‘-- H2N H2N H2N HO H 0 c á Pindolol Metoprolol M W = 2 Propranolol 4 8 MW=267 M W = 2 5 9 Parent Ion Scan Example: From a mixture, determine which components a r e from t h i s c l a s s of beta blockers MD(_MS 1 (1,025> F o r e x a m p l e , t h i s i s a MS S c a n S c a n ES+ 2 3 5 1.56e9 f r o m a mixture of components. 1001 2 6 8 280 Ii 2 6 7 260 2 4 9 % 2 9 5 I -0 m l z 230 235 240 245 250 255 260 265 270 275 280 285 290 295 300 T o determine which components belong t o t h i s c l a s s of b e t a blockers, you could p e r f o r m a daughter ion s c a n f o r daughter ions of t h e m/z=235 see p a r e n t ion a n d i f t h e m/z=235 ion p r o d u c e s a m / z = 116 daughter ion, t h e n a n o t h e r daughter i o n s c a n on m/z=249, t h e n 260, etc. Alternatively, you could do o n e p a r e n t ion s c a n f o r p a r e n t s of t h e m/z=116 daughter ion. 5 8 Quattro m i c r o Training Course

  44. Parent Ion Scan Example: MiX_MS 1(1.025) Scan ES+ MS Scan of Mixture of Components. 1.56e9 235 268 280 U 267 260 249 % 295 L MIX ‘AR 1 (2.070) Parents of 116ES+ 249 5.63e7 Metoprolol 100- Parent Ion Scan of Mixture: Propranolol Pindolol a %- Parent Ions of the 260 268 m/z= 116 Daughter Ion 0— 230 240 250 260 270 280 290 300 Parent Ion Scan 17-a Hydroxyprogesterone MW 330 17-OHP_DAU_001 1(0.527> Daughters of 331 ES* 97.1 2.49e6 100 Daughter Scan for Products 109.1 331.3 of miz 331 Ions from a % 17-a Hydroxy Progesterone 713 2 f. 25 Sample ii flI 5. ii I hil 3 75 100 125 150 175 200 225 250 275 300 325 350 375 400 ‘çrhere is a class of steriods whose molecular ions (M+H) produce the mlz97 and mlz=109 daughter ions when they undergo CID PARENT-MSMS-003 1 (1.103> Parents of 97AP* 3154 1.50e6 100 Progesterone 289.4 Parent Scan 17-a Hydroxy for Parents Progesterone Testosterone of mlz = 97 Ions % from a Mixture 331 5 of Steroids C 50 75 100 125 150 175 200 225 250 275 300 325 350 375 400 59 Quattro micro Training Course

  45. I I I Constant Neutral L o s s I Different Compounds Same Different That Are Somewhat Neutral Charged I Similar In Structure Fragment Fragments CID I -4 + I CID I -4 + I Constant Neutral Loss Scans can be used to detect those compounds whose molecular ions produce the same neutral fragment. I Constant Neutral L o s s ( C N L ) Scan MS1 Collision MS2 Cell (w/Argon) Scanning Scanning 5-40 eV m 1 -offset m 1 m 2 m 2 - offset Qi and Q2 scan together. m/z of Q2 is m/z of Qi minus an offset. 60 Quattro micro Training Course

  46. Tricyclic Antidepressants that All Produce a Daughter Ion After a CID Loss of m/z= 195 flNH* Charged Fragment NH Neutral Fragment Desipramine (MW=195) H Charged Fragment Neutral Fragment NH I (N W=1 95) Trimipramine CNL Example: From a mixture, determine which components are from this class of antidepressants MIX_MS 1(1.025) For example, this is a MS Scan Scan ES’- 100 From a mixture of components. 268 260 267 260 249 % 295 m/z r,., . . . 0 . . 230 235 240 245 250 255 260 265 270 275 280 285 290 295 300 To determine which components belong to this class of tricyclic anti- depressants, you could perform a daughter ion scan for daughter ions of the m/z=235 parent ion and see if the m/z=235 produces a daughter ion that is lighter by m/z= 195 (235-195=40), then repeat this with daughter ion scans of m/z=249, m/z=260, etc. Alternatively, you could do one Constant Neutral Loss scan for losses of 195. 61 Quattro micro Training Course

  47. Constant Neutral Loss S c a n Example M S Scan of M i x t u r e of Components. MIX M S 1 ( 1 0 2 Es+ 5 ) s c 2 3 5 1 . 5 6 e 9 2 6 8 2 8 0 M I X _ C N L 1 (2.017) Neutral Loss 195ES+ 295 5 . 0 1 e 7 100- C o n s t a n t Neutral L o s s Scan of Mixture 267 P a r e n t Ions which T r i m i p r a m i n p r o d u c e e D a u g h t e r I o n s _ ° m f z = 195 with a Loss of D e s i p r a m i n e f r o m t h e Parent I o n I I 230 240 2 5 0 260 270 2 8 0 2 9 0 3 0 0 C o n s t a n t N e u t r a l L o s s (CNL) Scan FENSO3 1(2017) Neu0( Lc 44ES- o 253 334e7 100 243 OH I - Flurbifen Ketoprofen / 205 I b u p r o f e n 163 ‘ “ 1 ’ ,1W2 160 170 180 190 200 210 220 230 240 250 260 270 280 Negative Electrospray Neutral Loss Scan for loss o f 4 4 m l z D a f r o m a M i x t u r e o f C o m p o u n d s = 6 2 Quattro m i c r o T r a i n i n g Course I

  48. Analysis of Amino Acids by MS/MS — CNL OH Phenylalanine 0 N 0 N’ HO Tyrosine Analysis of Amino Acids by MS/MS — CNL O 0 Deny — — / / “ “ mlz=222 Phenylalanine 0 0 Deny rN— Hoi HOcJ’ mlz = 238 Tyrosine 63 Quattro micro Training Course

  49. A n a l y s i s of Amino Acids by MSIMS C N L — OH 0IZ2220 Phenylalanine m l z 120 = C . Deny l . D . - - / HO “S HO HO mIz 238 ‘ Tyrosine m ! z 136 = CID R e s u l t s i n t h e L o s s 102 of D a I A n a l y s i s of Amino Acids by MSIMS CNL — D e r i v i t z e d Amino Acid Mix Infused 10 ijLlmin Quattro micro 1M NeoLynxTestMix_CNL 1(0.510) Neutral Loss 1O2ES+ 188.0 191.1 4.28e7 IULf \ Leucine Phenylalanine \ 222.1 T y r o s i n e / a,,. 227.2 o Methionine \ 23.12/40.1 209.0 206.1 0 I i mlz 180 185 190 195 200 205 210 215 220 225 230 235 240 245 250 O t h e r p e a k s are from deuterated forms of t h e s e a m i n o a c i d s 6 4 Quattro micro Training Course

  50. Phospholipids — Plant Extracts 100 MS Scan of Plant Extract . L J % J i U LrLrniz 400 500 600 700 800 900 1000 L-Phosphatidylcholine H 2 COOCR” (R” = H for lyso PC (PC and lyso PC) I or lyso PE) R’COOCH 0 L-Phosphatidylethanolamine ii (PE and lyso PE) + —CH — 2 2 C—O—P—0—CH 2 H N X 3 X = CH 3 for PC & lyso PC} X = H for PE and lyso PE Phospholipids — Precursor Ion Scan Example 100 MS Scan of Plant Extract % I L J , L m i z 0 400 500 600 700 800 900 1000 L-Phosphatidylcholine H 2 CO0CR” (R” = H for lyso PC) (PC and lyso PC) R’COOCH 0 Positive ion electrospray: II When PC and lysoPC ionize + H 2 C — 0 — P — 0 — CH 2 — CH 2 — N (CH 3 ) 3 and fragment at the red f arrow, the bottom right fragment leaves as a - charged ion with m/z=184. 65 Quattro micro Training Course

  51. Phospholipids — Precursor Ion Scan Example 100 MS Scan of ant Extract L J L,Limiz 4C 500 600 700 800 900 1000 100 MS/MS Precursor Scan for Precursors of m/z= 184 to detect 0/ 0 different forms of PC and lyso PC - 111 -#k urArr,. . 1 r T . mlz . 400 500 600 700 800 900 1000 Samples provided by Dr. Ruth Welti, Kansas State University, Manhattan, KS Phospholipids — Constant Neutral Loss Example 100 MS Scan of o: PntExtradiL, 400 500 600 700 800 900 1000 L-Phosphatidylethanolamine 2 COOCR” H (R” = H for lyso PE) (PE and lyso PE) R’COOCH 0 Positive ion electrospray: When PE and lyso PE ionize — p H c 2 — 0 — 0 — CH 2 — CH 2 — NH 3 and fragment at the red .f II arrow, the bottom right fragment leaves as a neutral I fragment of mass 141. 66 Quattro micro Training Course

  52. ________ ______________ Phospholipids — Constant Neutral Loss Example 100- MS Scan of MS Scan Plant Extract %- 0- 400 500 600 700 800 900 1000 100- MS/MS CNL Scan for Neutral Losses of 141 amu to detect %- different forms of PE and lyso PE i m/z Viiii 400 500 600 700 800 900 1000 Samples provided by Dr. Ruth Welti, Kansas State University, Manhattan, KS Phospholipids — Neutral Loss and Precursor Ion 100- MS Scan of MS Scan Plant Extract %- 0- 400 500 600 700 800 900 1000 <- PC and iyso PC by 100 Precursorlon Scan (Parent Ion Scan) L J OL PE and lyso PE by CNL -> 0 iIT)/Z .1-’.1.—, i’’i•• ‘ii 400 500 600 700 800 900 1000 Samples provided by Dr. Ruth Welti, Kansas State University, Manhattan, KS 67 Quattro micro Training Course

  53. M S S c a n of N a t u r a l Product P h a r m a c e u t i c a l TM Quattro m i c r o M S Example 1 (1.009) S c a n E S 349.1 100• 2.82e8 M S S c a n 3 4 7 350.0 345’ 297.1 269.1 365.1 379.1 .j. .,,, 1 rr1, ii 1, i 1 i J z 200 220 240 260 280 300 320 340 360 380 400 420 440 E x a m p l e of a M S S c a n of a natural product pharmaceutical. MS S c a n of N a t u r a l Product Pharmaceutical Quattro microTM MS Example 1 (1.009) Scan ES- 349.1 1 0 0 2.82e8 MS S c a n % 347.0 \ 0.0 297.1 269.1 365.13791 dl 9 3 0 0 21 0 220 240 260 280 320 340 360 380 400 420 440 C N L S c a n of N a t u r a l Product P h a r m a c e u t i c a l Quattro microTM C N L Example 1 (2.017) Neutral Loss 80E5- 349.1 4.36e7 C N L S c a n f o r l o s s of m l z = 8 0 . A c t i v e i n g r e d i e n t s i n mixture w o u l d e x h i b i t a loss of 8 0 . , a 350.2 345J / 3 6 5 . 2 0 I I i i I I Z i 200 220 240 260 280 300 320 340 360 380 400 420 440 6 8 Quattro m i c r o Training Course

  54. MS Scan of Natural Product Pharmaceutical Quattro micro MS_Example 1 (1.009> Scan ES 2.82e8 1oo.° 297.1 365.1 MS Scan 2691 345.0 Magnified %. lox 379.1 399.14j3.2 253.0 398.1 I 213.1 230.9 283.0 15.1 431.1 L di L I. •• - - - n - f l mlz mYTmrr1rrT rrrnmTmvTrrrrrrn-n . 21 0 220 240 260 280 300 340 320 360 380 400 420 440 CNL Scan of Natural Product Pharmaceutical Quattro microTM CNL_ Example 1 (2.017) Neutral Loss 8OES- ioo t(10 4.36e7 >51 .1 CNL Scan Magnified lOX. 36 5.2 Only those components in 345.1 the CNL Scan are active %. components. 366.1 23.1 IF hrrr, m/z - . I- .ir. . . .. 300 200 220 240 260 280 320 340 360 380 400 420 440 Waters Quattro Instrument Tune Page Quattro micro MICROMASS M r .0ClG 69 Quattro micro Training Course

  55. C C -I. - ‘ 0 3 C) - , 0 - m x D C, CO C, C-) CD 3 0 C’, c i , -‘ Cl) CD CD 0 0 Cn — —1= D) — C f l 3 -‘ (1) 00 C D Cl) -I - i C - 0 3 CD CD — — — — — — — — — — — — — — — — — — —

  56. ___________________________________ ___________________ Tune P a g e the b r a i n of t h e m a s s spec - Ion • mode is s e t Instrument parameters • a r e set. • API and Collision gases are controlled. • Parent compounds are identified and optimized • Daughter ions a r e determined and optimized • Mass Resolution is s e t Collision • Energies are determined • Acquisitions performed • P M T is adjusted when n e c e s s a r y I n s t r u m e n t Parameters Can B e ‘Saved’ in a File. Instrument tune page parameters can be stored in a file for future use. As with most MS Windows programs that save data in a file, the ‘Open’ (to load previously saved parameters back into the mass spectrometer), ‘Save’ (store current instrument parameters) and ‘Save As’ (save the parameters under a different file name) features are available along with ‘Print’ capabilities. ur i I *F Dlxi i i ’ ti i i [Thit’! I]IIk?1111 ‘r—$ *4 ‘ EeiMoaeaiib a a r p p b o n s fielpT, j I il1!i1 m I. 1.-I I ‘ ‘ Use the ‘New’, ‘Open’, ‘Save’ or ‘Print’ buttons or use the ‘File’ menu for these operations. 71 Quattro micro Training Course

  57. _____________________________ ____________ ________ ___ ____ ___ Current Ion Mode of the Mass Spectrometer is Operating is Shown on the Front Tab In the example shown below, the mass spectrometer is operating in the positive ion electrospray mode. IQuattro Micro - c:massIynx\1raininq.pro\acq. RLIE Ee !onI4oc aIrbibn n: Raptins Hel DI1I1 - Alaly:er I Diagnostics [ [s+ Source ‘-Voltages—--- j I I H 1356 1i0 rRI1III rkvl The Ion Mode Can be Changed Electr’ ‘S Using the Electropray APcl+ ‘Ion Mode’ Menu El - l ’ A - Nitrogen Gas and Collision Gas (Ar) These Gases I Quattio Micro - c:massIynx\Lrainingpro\acq I J ! Can be File Ion Mode Calibiation Gas Ramps Options Help ToggledOn -- ---•-• — --—--- - i1I1Fiij1T1 t?I1Il DIIIII andOffUsing Buttons or E5+ Source Tune Page I ‘--Voltaaes Menu Items —-- --- — I DI XJ - I fleip naiiq uun lull IVlUUO L110 .,IIUIdLIU1 ‘Gas I 1 1 1 : J El i,’Collisjonas I ES+ Source Ana!yser Inject Voltages-— .-—--.-...-..--—--- I —J--. Gas Fail Override capillary [kV) — I 72 Quattro micro Training Course I

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