Maximizing MS/MS Fragmentation in the Ion Trap Using CID Voltage ...

Maximizing MS/MS Fragmentationin the Ion Trap Using CID VoltageRampingTechnical NotePaul GoodleyAgilent TechnologiesIntroductionA seldom-discussed obstacle to obtaining good,consistent MS/MS results with triple-quadrupoleand ion-trap mass spectrometers is the compounddependentnature of collision optimization. Collisiongaspressure and collision energy both have a significanteffect on sensitivity, mass spectral quality, andion yield. In chromatographic analyses with multipleanalytes, it can be difficult or impossible to find asingle set of collision parameters that yield optimumproduct ion spectra for all analytes.This note discusses the effect of these collisionparameters in triple-quadrupole and ion-trap massspectrometers. It shows how the new SmartFragfeature in the Agilent 1100 Series LC/MSD Trapion-trap mass spectrometer overcomes compounddependency to generate optimum information fromevery analyte.Triple-quadrupole mass spectrometersyield useful fragment ions—at a priceIn a triple-quadrupole mass spectrometer, andsimilar quadrupole/quadrupole/time-of-flight instruments,the precursor ion is selected by the firstquadrupole and collision takes place in the collisioncell (second quadrupole). The collision-induceddissociation (CID) is “slow” because it takes placealong the entire length of the collision cell. In onesense this is an advantage, because the slow natureof this process allows cascading fragmentation totake place. Product ions generated by collision ofthe precursor ion are themselves fragmented byfurther collisions. The result is a wide range ofproduct ions that are useful in compound identificationand structure elucidation.There are, however, several corresponding disadvantages.In a triple-quadrupole mass spectrometer, bothcollision-gas pressure and collision energy, which

Maximizing MS/MS Fragmentation in the Ion Trap Using CID Voltage RampingAgilent Technologiesis controlled by the collision-cell voltage, havesignificant effects on analytical results. Both mustbe optimized in order to achieve the best analyticalresults. Because optimum settings are compounddependent, settings that produce good results forthe first compound to elute may yield poor resultsfor the next compound. A triple-quadrupole massspectrometer must be able to change settings foreach analyte to produce optimum results.Traditional ion traps havea different set of challengesThe ion trap works somewhat differently. Theprecursor ions are held near the center of the trapin a confined space. The collision time tends to beshorter (faster), allowing little or no time for furtherfragmentation of the product ions. The result isfewer product ions and less structural information.Traditionally, this was compensated for by usingthe ion trap’s ability to perform more levels ofMS analysis to continue selecting and fragmentingproduct ions. Indeed, this is a very powerfultechnique that allows structure to be determinedin a very controlled fashion. Recent innovations,however, allow the Agilent ion trap to generatemore structural information with fewer steps ofMS and at the same time overcome the compounddependentnature of collision optimization.In the ion trap, the collision gas, generally helium,reduces the kinetic energy of the incoming ions asthey approach the center of the trap. The amountof gas in the trap is generally fixed and is notchanged for different compounds. This leaves onevariable, collision energy, with a significant andcompound-dependent influence over fragmentation.CID voltage ramping in the ion trapprovides the best of both worldsA new feature, CID voltage ramping (SmartFrag),is built into firmware of the Agilent 1100 SeriesLC/MSD Trap. CID voltage ramping eliminates theneed to optimize the collision energy for differentcompounds. Each time MS/MS is performed, CIDvoltage ramping quickly ramps the collision voltageover a range of energies. This ensures that eachcompound receives the correct energy for optimumfragmentation. It also results in a higher level offragmentation, providing more structural informationwith fewer steps of MS/MS.Figure 1a shows the full-scan mass spectrumof prednisone, an anti-inflammatory steroid havinga protonated molecular ion [M+H] + at m/z 359.Figure 1b depicts the MS/MS full-scan spectrumobtained at a fixed collision energy of 0.8v. TheMS/MS spectrum yields a large precusor ion anda few product ions which show m/z losses for awater molecule and for a water molecule and CO(total of 46). Figure 1c shows the same MS/MSfull-scan spectrum obtained using the CID voltageramping. The spectrum is rich with product ionsproviding much structural information.2

Maximizing MS/MS Fragmentation in the Ion Trap Using CID Voltage RampingAgilent TechnologiesIntens× 10 6 Ion-Trap Full-Scan MS of1.5 Prednisone; Molecular Ionat m/z 359OCH 3CH 2 OHCH 3OOH+ All MS, 3.0 min (#2)359.11.0O[M + H] +a0.50.0200 220 240 260 280 300 320 340 360m/zIntens+ MS/MS (359), 0.9 min (#46)× 10 6 Full-Scan CID MS/MS ofO359.1m/z 359; Non-rampedCH 2 OHCH 31.0 CID VoltageOOH0.8CH 30.6O0.40.2313.2341.10.0200 220 240 260 280 300 320 340 360 m/z×Intens10 52.5+ MS/MS (359), 1.3 min (#5)Full-Scan CID MS/MS ofO CH 2 OH341.1CHm/z 359; using Ramped3OOHCID VoltageCH 32.0313.01.5O323.0295.01.0305.00.5212.9267.0186.9237.1 253.1 277.0287.00.0200 220 240 260 280 300 320 340 360 m/z[M + H – 46] +[M + H – 2 (H 2 0)] +[M + H – H 2 0] +[M + H – H 2 0] +[M + H] +bcFigure 1. MS/MS of Prednisone using CID voltage ramping3

Maximizing MS/MS Fragmentation in the Ion Trap Using CID Voltage RampingAgilent TechnologiesFigure 2a is a full-scan mass spectrum of theantihypertensive, Labetalol, having a protonatedmolecular ion at m/z 329. Figure 2b shows MS/MSof the precursor ion yielding a major ion at m/z 311,which corresponds to the loss of a water molecule.The spectrum is nearly bare of product ions, presentingthe need for more stages of MS to more fullyconfirm the molecular structure. If this occurs witha triple-quadrupole system, the option for MS 3 doesnot exist and other confirmatory approaches may beneeded. Figure 2c shows MS/MS of Labetalol usingCID voltage ramping. The full-scan MS/MS production spectrum is rich with product ions and the mostabundant ion in the spectrum is at m/z 207. Thisprovides much more structural information than thesingle ion at m/z 311.Intens× 10 5 Ion-Trap Full-Scan MSYields Molecular Ion6 at m/z 3294H 2 NHOOOHHNCH 3+ All MS, 1.4 min (#54)329.2[M + H] +a2Labetalol0100 125 150 175 200 225 250 275 300 325 m/zIntens× 10 51.51.00.50.0Intens× 10 43210Classical Ion Trap WithoutCID Voltage RampingFull-Scan CID MS/MSOYields Major IonHas Loss of Water2 Nat m/z 311HOFull-Scan CID MS/MSof m/z 329 using CIDVoltage Ramping147.1162.1179.1190.1207.1207.1H 2 N100 125 150 175 200 225 250 275 300 325 m/zHOOOHHN266.1+ MS/MS (329/311), 2.9 min (#84)311.2100 125 150 175 200 225 250 275 300 325 m/zOHHNCH 3CH 3294.2+ MS/MS (329/311), 3.3 min294.2[M + H – H 2 0] +311.2[M + H – H 2 0] +bcFigure 2. MS/MS of Labetalol using CID voltage ramping4

Maximizing MS/MS Fragmentation in the Ion Trap Using CID Voltage RampingAgilent TechnologiesFigure 3 is another example of the use of CIDvoltage ramping. Figure 3a is the full-scan massspectrum of 4-hydroxytamoxifen, a compound forthe treatment of breast cancer. Figure 3b showsresult of MS/MS of tamoxifen at a fixed collisionvoltage of 0.9 V. Although the spectrum yields sixto seven ions useful for structural characterization,the precusor ion is not totally fragmented. Furtheranalysis would be required to find the optimumCID voltage to obtain a more spectral-rich production spectrum. Without other experimental changes,switching to CID voltage ramping generated theproduct ion spectrum shown in Figure 3c.Intens× 10 6 Ion-Trap Full-Scan MS5 Yields Molecular Ionat m/z 388432CH 3OHONCH 3CH 3+ MS/MS (388), 0.8 min388.2[M + H] +a14-Hydroxytamoxifen03210100 150 200 250 300 350 400 m/zIntens× 10 5 Full-Scan CID MS/MS4 of m/z 388; Non-rampedCID voltage128.9 144.9166.1209.1223.1249.1+ MS/MS (388), 5.3 min100 150 200 250 300 350 400 m/zFull-Scan CID MS/MSIntens of m/z 388 using CID× 10 4 Voltage Ramping3210166.1183.1223.1CH 3128.9 145.0249.1194.1OHCH 3265.1287.1OOH287.1 301.2100 150 200 250 300 350 400 m/zN316.2316.2CH 3CH 3343.2O343.2[M + H – 45] +NCH 3CH 3388.2[M + H] ++ MS/MS (388), 2.5 min[M + H – 45] +bcFigure 3. MS/MS of 4-hydroxytamoxifen using CID voltage ramping5

Maximizing MS/MS Fragmentation in the Ion Trap Using CID Voltage RampingAgilent TechnologiesConclusionsUsing the CID voltage ramping feature of theAgilent LC/MSD Trap ion-trap mass spectrometereliminates the need for time-consuming collisionvoltageoptimization. By setting a range of CIDvoltages using the SmartFrag setting, an analystcan be certain to obtain fragment-rich, full-scanproduct-ion spectra for identity confirmation orstructure elucidation.For more information on our products and services, you can visit our siteon the World Wide Web at: http://www.agilent.com/chemAgilent Technologies shall not be liable for errors contained herein or forincidental or consequential damages in connection with the furnishing,performance or use of this material.Information, descriptions and specifications in this publication aresubject to change without notice.Copyright © 2000Agilent TechnologiesAll rights reserved.Reproduction and adaptation is prohibited.Printed in the U.S.A. November 2000(23) 5988-0704EN