Ultra High Definition Optimizing all Analytical Dimensions Sensitivity Dynamic Range Signal Response Linearity Separation Speed Peak Capacity Chromatogram Mass Spectrum Mass Accuracy Resolving Power Acquisition Rate ASMS 9 UHPLC 654 QTOF lunch and learn Page June 9
Agilent s New 654 Ultra High Definition Accurate Mass Q-TOF Exceptional accurate mass, sensitivity, dynamic range and resolution perfect match for 9 Infinity UHPLC 5 ppb mass accuracy femtogram sensitivity 5 decades dynamic range 4, resolving power Spectra/sec Excellent Linearity and Isotopic Fidelity Supports Agilent Jet Stream and HPLC-Chip ASMS 9 UHPLC 654 QTOF lunch and learn Page June 9
Ion Beam Compression (IBC)* Technology Drives Higher Resolution Compressed and cooled ion beam ensures the best sensitivity performance in high resolution mode Exit from collision cell Into slicer and pulser region Narrowed beam slit enables mass resolution of 4K Up to -fold ion beam compression * Patent pending ASMS 9 UHPLC 654 QTOF lunch and learn Page 3 June 9
654 Ultra High Definition QTOF Maintaining Resolving Power Across the Mass Range 6.8 m/z 6 and isotopes 5 x 5.5 5 4.5 4 6 R=387 9 R=444 R=43674 63.59 64.9 65.47 3.5 5 3.5.5 3 R=38 R=475 8 R=445 R=485 4 R=3933 7 R=377.5 4 6 8 4 6 8 4 6 8 Scan Rate Independent ASMS 9 UHPLC 654 QTOF lunch and learn Page 4 Orbitrap evaluation March June 9
654 Ultra High Definition QTOF Mass Accuracy Repetitive Injections 4pg reserpine on-column, injections +ESI EIC(69.866) Scan Frag=4.V Reserpine_4pgms3.d.5..5.5.75 Counts vs. Acquisition Time (min) Error (ppm) Run.96.7 3.5 4. 5.39 6.3 7. 8.5 9.4.3 Mean.5 Std. Dev.3 5 x..8.6.4. Isotope 69.88 Obs % Calc % Obs m/z Calc m/z Diff (ppm) 69.88 69.866.5 35.87 37.4 6.845 6.8393.5 3 8.58 8.59 6.865 6.867 -.34 4.4.48 6.8987 6.894.75 5.4. 63.9 63.93. 6.845 6.865 6.8987 63.9 69 6 6 6 63 64 5 ppb mass accuracy calibration and very accurate isotopic ratios ASMS 9 UHPLC 654 QTOF lunch and learn Page 5 June 9
654 Ultra High Definition QTOF Sensitivity Full Scan MS Mode- picogram 4 x S/N = 39 RMS Excellent sensitivity, accuracy and resolution at pg on column 3 x Resolution ~ 33, 69.8347 Ratio m/z Diff. Theor. Expt. Theor. Expt. (ppm).. 69.87 69.83.5 37.4 36.36 6.839 6.83.5 8.59 8.39 6.867 6.878.77.48.45 6.894 6.96 5.9 6.8368 6.8779 6.9649..5.5 Counts vs. Acquisition Time (min) 67 68 69 6 6 6 63 64 65 66 ASMS 9 UHPLC 654 QTOF lunch and learn Page 6 June 9
Topics Example: Pesticides in Foods Example: Drug metabolite identification ASMS 9 UHPLC 654 QTOF lunch and learn Page 7 June 9
Pesticides Screening In Foods Accurate-Mass Databases for Comprehensive Screening of Pesticide Residues in Food by Fast Liquid Chromatography Time-of-Flight Mass Spectrometry, Milagros Mezcua, Octavio Malato, Juan F. García-Reyes, Antonio Molina-Díaz, and Amadeo R. Fernández-Alba, Anal. Chem., 9, 8 (3), 93-99 DOI:./ac84t Method for screening 97 pesticides in foods with Agilent HPLC, 4.6 x 5 mm XDB-C8,.8 µm column, and TOF-MS detection Component identification using accurate mass/retention time (AMRT) matching against a database.6 ml/min flow rate, minute gradient, approximately 3 minutes run time Comparable method performance when tested on 6 commercial produce samples (QuEChERS protocol sample prep) and compared with results from QQQ [note: 45/6 samples tested positive!] Identified several pairs of isobaric coeluting species (ICS) which have similar retention times and accurate mass values for their adduct ions or fragments ASMS 9 UHPLC 654 QTOF lunch and learn Page 8 June 9
Experimental Conditions For Pesticides Isobaric Coeluting Species Analysis Configuration 9 Infinity UHPLC, 654 QTOF with Agilent jet stream technology Eclipse Plus C8 column,. x 5 mm or mm,.8 µm Analysis conditions A =.% formic acid in water, B =.% formic acid in ACN; various flow rates (.,.,.5 ml/min); 6 ºC column temperature Gradient = 5% B to 95% B in various times (.5, 3, 7, 8.5, 7 minutes) 3 m/z maximum mass, high resolution mode MS mode: - m/z, various acquisition rates (, 5, 3, scans/second) ASMS 9 UHPLC 654 QTOF lunch and learn Page 9 June 9
RRLC UHPLC x 6.5 ml/min. x mm column, 6 ºC, max pressure 4 bar x 5.5. ml/min. x mm column, 6 ºC, max pressure 78 bar x 5. ml/min. x mm column, 6 ºC, max pressure 93 bar x 5.5. ml/min. x 5 mm column, 6 ºC, max pressure 5 bar 3 4 5 6 7 8 9 3 4 5 6 Counts vs. Acquisition Time (min) ASMS 9 UHPLC 654 QTOF lunch and learn Page June 9
Mass Accuracy And Mass Resolution And Analysis Speed Simultaneously x 5.5.5 x 6.5 3.498 spectra/second 3 4 5 6 7 8 9 3 4 5 6 Counts vs. Acquisition Time (min) spectrum/second.5.5 x 4.7 44.387.6.5.4.3...9.8.7.6.5.4.3.. -.6 ppm R = 3,6 45.667 O H 3 C CH 3 O O O 46.997 47.36 4 4.5 43 43.5 44 44.5 45 45.5 46 46.5 47 47.5 48 N N O azoxystrobin N 8.7 3 4 5 6 7 8 9 3 4 5 6 Counts vs. Acquisition Time (min) x 5.7 44.395.6.5.4.3...9.8.7.6.5.4.3.. 45.694 -.37 ppm R = 3,4 46.956 47.39 48.3 4 4.5 43 43.5 44 44.5 45 45.5 46 46.5 47 47.5 48 Page ASMS 9 UHPLC 654 QTOF lunch and learn June 9
Mass Accuracy Is Invariant With Acquisition Rate Pesticides Example ASMS 9 UHPLC 654 QTOF lunch and learn Page June 9
MS Resolution Is Invariant With Acquisition Rate Pesticides Example ASMS 9 UHPLC 654 QTOF lunch and learn Page 3 June 9
Isobaric Coeluting Species (ICS) Definitions Isobaric pairs whose diagnostic ions differ in mass by between. Da and. Da Formula m/z Compound ppm difference C9H7N3S 9.433 a tricyclazole C9H6ClN5 9.667 b propazine F Cl 37 3 C4HNO4 6.74 a diethofencarb F C4H5N3 6.339 b cyprodinil 7 C5H8ClN3O 94.8 3a cyproconazole Cl 37 C5HClN3O 94.368 3b paclobutrazol 63 C3HClF4N3O 37.88 4a tetraconazole CH3N3O4 37.979 4b Azoxystrobin F 86 Co-eluting not clear, but pairs whose retention times differ by less than approximately. minutes under the analysis conditions (-3x peak width at half height)
Group : Identify Them By Differences In RT x 4 8 7 55.7 Propazine (fragment) 6 5 4 3 Tricyclazole 39. 5 5 5 3 35 4 45 5 55 6 65 7 75 8 85 Counts vs. Acquisition Time (sec) ASMS 9 UHPLC 654 QTOF lunch and learn Page 5 June 9
Group : Diethocarb Fragment And Cyprodinil Identify By Difference In Accurate Mass EICs Hypothetical complete coelution (average spectrum) x 5..5.95.9.85.8.75.7.65.6.55.5.45.4.35.3.5..5..5 cyprodinil 56. 57. diethofencarb fragment 5 5.5 53 53.5 54 54.5 55 55.5 56 56.5 57 57.5 58 58.5 59 59.5 6 6.5 Counts vs. Acquisition Time (sec) x 4.7.6.5.4.3...9.8.7.6.5.4.3...9.8.7.6.5.4.3.. diethofencarb fragment -.99 ppm error 6 mda difference (7 ppm) 6.75 6.33883 Cyprodinil -.3 ppm error 6.6 6.8 6. 6. 6.4 6.6 6.8 6. ASMS 9 UHPLC 654 QTOF lunch and learn Page 6 June 9
ICS Group 3: Identify These Pesticides By RT And Accurate Mass x 4.8.6.4..8.6.4.. By retention time paclobutrazol 4.357 cyproconazole- 37 Cl 4.43 4.457 4.8 4. 4. 4.4 4.6 4.8 4.3 4.3 4.34 4.36 4.38 4.4 4.4 4.44 4.46 4.48 4.5 4.5 4.54 4.56 Counts vs. Acquisition Time (min) N N N OH CH 3 H Cl N N N H CH 3 OH Cl x 4.4. By accurate mass value 94.3659..8.6.4. of the monoisotopic peak cyproconazole- 37 Cl Cyproconazole- -.85 ppm 37 Cl R = 468 94.79 8 mda separation (63 ppm) paclobutrazol paclobutrazol -.63 ppm R = 76 94 94. 94.4 94.6 94.8 94. 94. 94.4 94.6 94.8 94. 94. ASMS 9 UHPLC 654 QTOF lunch and learn Page 7 June 9
ICS Group 3: Average The Two Peaks Into A Single Spectrum x 3.6 94.36473.4. Paclobutrazol -.99 ppm.8.6.4..8.6.4. Cyproconazole- 37 Cl -.59 ppm 94.7994 8 mda separation (63 ppm) 94.7 94.8 94.9 94. 94. 94. 94.3 94.4 94.5 94.6 94.7 94.8 94.9 94. 94. ASMS 9 UHPLC 654 QTOF lunch and learn Page 8 June 9
ICS Group 3: Identify These Pesticides By Isotopic Pattern And Mass x 3 3.5 3.5 cyproconazole 9.9.5.5 94.79 93.384 95.4 89 9 9 9 93 94 95 96 97 98 99 3 3 x 4..8 paclobutrazol 94.3659.6.4. 96.3354 95.3984 97.379 98.397 89 9 9 9 93 94 95 96 97 98 99 3 3 ASMS 9 UHPLC 654 QTOF lunch and learn Page 9 June 9
ICS Group 4: Identify Them By Differences In RT Or Accurate Mass x 3 9 9. 8. 7 6 5 4 3. By retention time Azoxystrobin (fragment) 84.6. Tetraconazole PWHM ~. sec 55 6 65 7 75 8 85 9 95 3 35 3 35 3 35 Counts vs. Acquisition Time (sec) x 3 6 5 4 3. By accurate mass value of the monoisotopic peak Tetraconazole 37.85 R = 8458 -.8 ppm 37.977 R = 8394 -.54 ppm 69 mda separation (86 ppm) Azoxystrobin (fragment) 37.85 37.9 37.95 37 37.5 37. 37.5 37. 37.5 37.3 ASMS 9 UHPLC 654 QTOF lunch and learn Page June 9
ICS Group 4: Identify Them By Differences In Isotopic Pattern x 3 8 7 6 5 4 3 Azoxystrobin (fragment) 37.977 373.4 374.34 37.5 37 37.5 37 37.5 373 373.5 374 374.5 375 375.5 376 376.5 377 x 3 8 Tetraconazole 37.85 7 6 5 4 3 373.34 374.57 375.84 376.6 37.5 37 37.5 37 37.5 373 373.5 374 374.5 375 375.5 376 376.5 377 ASMS 9 UHPLC 654 QTOF lunch and learn Page June 9
ICS Summary Formula m/z Compound ppm difference Identify by C9H7N3S 9.433 a tricyclazole C9H6ClN5 9.667 b propazine F Cl 37 3 RT difference C4HNO4 6.74 a diethofencarb F C4H5N3 6.339 b cyprodinil 7 MS difference C5H8ClN3O 94.8 3a cyproconazole Cl 37 C5HClN3O 94.368 3b paclobutrazol 63 MS difference C3HClF4N3O 37.88 4a tetraconazole CH3N3O4 37.979 4b Azoxystrobin F 86 MS difference ASMS 9 UHPLC 654 QTOF lunch and learn Page June 9
Final Fast Analysis: ICS ( ppb) In Red Pepper Matrix x 6.4.3...9.8.7.6.5.4.3... x 5 mm Eclipse Plus C8. ml/min, 6 ºC 4 6 8 4 6 8 4 6 8 3 3 34 36 38 4 4 44 46 48 5 5 54 56 58 6 6 64 66 68 7 7 74 76 78 8 8 84 86 88 Counts vs. Acquisition Time (sec) Name RT Mass Mass (Tgt) Diff (Tgt, ppm) Score (Tgt) a tricyclazole.65 89.3675 89.3668.57 98. b propazine.99 9.9356 9.943 -.9 97.5 a diethofencarb.953 67.46937 67.4758 -.45 85.49 b cyprodinil.936 5.66 5.6598. 98.8 3a cyproconazole.95 9.46 9.384.9 97.9 3b paclobutrazol.93 93.9448 93.949 -.4 96.74 4a tetraconazole.97 37.9 37.53 -.65 99.45 4b azoxystrobin.957 43.6637 43.68 -.45 99.9 a 5 pg on-column x 5.9.8.7.6.5.4.3.. 3b b 3a b a 53.5 54 54.5 55 55.5 56 56.5 57 57.5 58 58.5 59 Counts vs. Acquisition Time (sec) 4b 4a 4 6 8 4 6 8 4 6 8 3 3 34 36 38 4 4 44 46 48 5 5 54 56 58 6 6 64 66 68 7 7 74 76 78 8 8 84 86 88 Counts vs. Acquisition Time (sec) ASMS 9 UHPLC 654 QTOF lunch and learn Page 3 June 9
Experimental Conditions For Verapamil Metabolite ID Analysis Configuration 9 Infinity UHPLC, 654 QTOF with Agilent jet stream technology Eclipse Plus C8 column,. x 5 mm,.8 µm Analysis conditions A =.% formic acid in water, B =.% formic acid in ACN;. ml/min; 6 ºC column temperature Gradient = 5% B to 95% B in. minute, stoprun.5 minutes, postrun.5 minutes 7 m/z maximum mass, extended dynamic range mode MS mode: - m/z, 3 scans/second ASMS 9 UHPLC 654 QTOF lunch and learn Page 4 June 9
Verapamil: Major And Minor Metabolites (Phase I) x 6 TIC verapamil x 5 5 desmethyl x 4 5.5 x 5 monohydroxy dihydroxy x 3 Isopropyl dealkylation..5..5.3.35.4.45.5.55.6.65.7.75.8.85.9.95.5..5..5.3.35.4.45 Counts vs. Acquisition Time (min) ASMS 9 UHPLC 654 QTOF lunch and learn Page 5 June 9
Coeluting Metabolites With Parent Drug: Need Wide Dynamic Range Five decades of response in a single scan verapamil dihydroxy metabolite of verapamil 6 x.8 455.93.6 counts.4..8.6.4..8.6.4..68 million desmethyl metabolite 44.7474 monohydroxy metabolite 47.8487 487.798 435 44 445 45 455 46 465 47 475 48 485 49 495 5 55 x 4 3.5 3.5.5.5 4 counts 487.798 488.834 5 counts 489.9744 49.8383 486.8585 489.848653 486.5 487 487.5 488 488.5 489 489.5 49 49.5 ASMS 9 UHPLC 654 QTOF lunch and learn Page 6 June 9
Metabolite ID Results ASMS 9 UHPLC 654 QTOF lunch and learn Page 7 June 9
Summary Unmatched chromatographic peak capacity and resolving power can even resolve diastereomers when parameters are adjusted appropriately Superb MS mass accuracy and resolution invariant with acquisition rate Sophisticated identification software and algorithms handle complex samples, overlapping isotopic patterns Sum (product?) is greater than the parts Run faster chromatography for higher throughput MS can keep up without sacrificing resolution MS can keep up without sacrificing mass accuracy MS has the dynamic range to handle coeluting peaks Software has the power to identify components of complex chromatograms and spectra Result: more information and results per analysis and per unit time