Impurity Identification using a Quadrupole - Time of Flight Mass Spectrometer QTOF

Impurity Identification using a Quadrupole - Time of Flight Mass Spectrometer QTOF

PUSHER TOF DETECTOR ZSPRAY TM Ion Source SAMPLING CONE SKIMMER RF HEXAPOLE RF HEXAPOLE QUADRUPOLE IN NARROW BANDPASS MODE HEXAPOLE GAS COLLISION CELL Q - TOF MS/MS MODE REFLECTRON Q-Tof Features Efficient duty cycle for enhanced sensitivity Mass range Acquisition of 1 scans/sec Resolution >5 FWHM <5ppm mass measurement (MS) <5ppm mass measurement (MS/MS) Automated MS-MS/MS switching

Typical Resolution achieved for accurate mass work peg12 115 (2.152) Cm (14:115) 68.3876 2516 Resolution = 62 69.436 84 61.3913 173 m/z 67 68 69 61 611 612 613 Calibration Report Fifth order best fit polynomial over 2-18 Da range Data file peg13 - Uncalibrated 697.95 653.85 742.6 38 matches of 38 tested references 83.27 874.37 416.25 54.47 962.57 15.77 1138.97 1271.26 1359.46 221.75 Residuals.1 Mean residual = 2.73e-8 ±.1311 amu. 2 3 4 5 6 7 8 9 1 12 13 14 15 16 17 18 19 2 M/z

Single Point Lock Mass Correction mass = A + Bt DRIFT mass = A + B 1 t A is unchanged so a single point lock mass enables B 1 to be calculated Why Exact Mass? Determination of elemental composition molecular ion and fragments Differentiation of nominal isobars combinatorial libraries Patent support and scientific journals 5ppm accuracy required Efficient database searching proteome elucidation

Measurement of ppm True mass = 4. Measured mass = 4.2 Difference =.2 2 mmu.2 ppm error = x 4 16 6 = 5 ppm The Experiment

Impurity Analysis Impurities in manufactured compound LC-MS analysis Exact mass measurement LC-MS/MS information Compound identification LC Conditions HPLC Waters Alliance Column C8 Flow 4uL/min Solvent 6:4 ACN/H 2 O +.1 formic acid Int ref Met Enkephalin (1ng/uL, 5uL/min)

MS Conditions Ion mode Electrospray + Cone 3V Internal ref Met Enkephalin (m/z 574.2771) Reference compound- Met. Enk. 5µL/min HPLC (Gradient) 4 2 µl/min Analytical column 2.1 x mm Q-Tof MS

2.18 11.5 4.42 5.83 13.43 Diode array 11.49 13.44 TIC 9.73 39 5. 5.72 7.23.2.88 1.57 6.24 A B C 11.57 D 13.44 m/z 519. 2. 4. 6. 8. 1. 12. 14. 16. Time LC-MS Results Mass chromatogram at m/z 519 Showed 4 components at nominal mass 518 Need to identify these impurities

Spectra of the 4 Components Theoretical monoisotopic [M+H] + = 519.1767 2.5ppm 519.178 52.185 Peak A 519.1776 1.7ppm 52.185 Peak B 519.1774 1.3ppm 52.1842 Peak C -.4ppm 519.1765 Peak D 52.1781 m/z 516 517 518 519 52 521 522 523 524 LC-MS Results Same exact mass for each component Agree to within 5ppm error Compounds are isomeric Need further information to identify Use LC-MS/MS

Data Dependent MS to MS/MS Instrument switched automatically to MS/MS Acquires product ion spectra Collision energy: 5, 1, 2, 3 and 4 ev Collision gas: argon Threshold: 5 counts/sec Product Ion Spectra for 4 Components 179.733 Peak A 487.1533 519.1767 52.1733 Peak B 487.1522 519.1767 488.158 x4 519.1767 487.1512 353.1147 Peak C 179.72 52.1754 487.1523 519.1767 179.719 Peak D 52.183 16 18 2 22 24 26 28 3 32 34 36 38 4 42 44 46 48 5 52 54 56 m/z

LC-MS/MS Results - Compound C Exact mass measurement Use precursor as lock mass Elemental composition of fragments Allowed structural elucidation at.3 level Structure of Peak C OMe m/z 353.1137 O OMe OMe R O OMe m/z 179.78

Conclusions Q-Tof sensitivity required for detection of impurities at.1 level Exact mass measurement determined that impurities had same elemental composition Exact mass MS/MS allows elemental composition of fragments to be determined NMR required for other impurities Considerations Lock mass may suppress ionisation of analyte (and vice versa). Solvent gradient effects may interfere with post column addition of lock mass compound. Lock mass contributes to TIC signal and may mask smaller analyte components.

LC-MS with Lock Spray Syringe pump Lock Spray LCT / QTOF Solvent Delivery System

Lock Spray LockSpray allows one conventional (or microbore) HPLC column to be interfaced in parallel with a second liquid inlet for the introduction of a mass reference standard. Compatible with both isocratic and gradient LC The oatof-ms automatically monitors the 2 separate electrospray inlets. A sampling rotor within the ion source (propelled by a programmable stepping motor) allows the 2 electrosprays to be sampled exclusively in rapid succession. The position of the sampling rotor is monitored in realtime enabling the two liquid inlets to be indexed Conclusions LC/MS and MS/MS can be performed in a single run Accurate mass measurements can be made on parent and daughter ions to suggest elemental composition Full scan sensitivity comparable to SIM sensitivity on quad systems