Q-TOF Lo strumento piu’ potente per l’identificazione di incogniti Maggiore selettività e riduzione del rumore Frammenti in Massa Accurata Self explanitory.

Primo quadrupolo in SIM! Come funziona un QTOF? Primo quadrupolo in SIM! HED Detector Quad Mass Filter (Q3) Quad Mass Filter (Q1) Quad Mass Filter (Q1) m2 m1 m4 m3 a Collision Cell Collision Cell Lens 1 and 2 Lens 1 and 2 Spettri in MSMS ACCURATA!

Diphenhydramine con Q-TOF 2 4 6 8 10 12 14 16 500 1000 1500 2000 2500 3000 3500 4000 4500 5000 5500 16.27 13.83 9.20 11.43 6.99 4.95 2.84 STD ESI: 0.25 mL/min flow Q1=256 (2.5 wide), CE=10v, 167 (0.1 wide) EIC 1000 fg 2000 fg 500 fg 200 fg 60 80 100 120 140 160 180 200 220 240 260 50 150 250 300 350 400 450 499 167.0805 257.1350 168.0836 179.0656 71.0826 117.0875 152.0602 MS/MS spectrum at 1 pg. Background substracted

Tabella comparativa degli strumenti Qualitativa Sensibilità SIM (campione semplice) Sensibilità in SIM (campione complesso) Sensibilità in SCAN (campione semplice) Sensibilità in SCAN (campione complesso) Quadrupolo 6 10 7 5 TOF 8 n/a (7) Ion Trap 9 n/a (8) Triplo Quadrupolo Q-TOF n/a (6) Sequenziamento di peptidi Note: this table is for internal distribution only. The intent of this table is to assist the salesperson in communicating the relative positioning of instrumentation in the LCMS portfolio. The customers we can best influence using this information are those who are new to LCMS. We want to be especially careful to not offend those experienced customers who have their own opinions. Furthermore, we don’t want the presentation to end up in the hands of our competitors, lest they try to use it against us. Ease of Use: learning curves are a combination of the user’s background and the level of difficulty in working with a particular instrument. We assume that for those customers who have overcome this learning curve and are comfortable with their day-to-day operation, the ease of use is then a function of how many parameters must be explored and optimized for method development. But ease of use may be a bigger issue for a lab with no LCMS experience than for labs which already have some form of LCMS. Scan speed: how long does it take to acquire a full scan MS spectrum for the single quad and TOF, and full scan MS/MS spectrum for the Ion Trap, QQQ and QTOF? Scan sensitivity: how much material is required to obtain a useful full-scan (not MS/MS) spectrum? Along with scan speed, does the instrument perform MS/MS, which is more sensitive for detection, or only MS, which is less sensitive? Qualitative: this ranking depends on how much you know about what you are looking for. A complete unknown may be better identified with accurate mass (empirical formula); a metabolite may be better identified with MS(n) (structure elucidation plus molecular weight). Sensitivity: signal-to-noise using the instrument in its best mode of operation. For an SQ: SIM; QQQ: SRM or MRM; TOF: full scan MS; Ion Trap: full scan MS/MS; and QTOF: full scan MS/MS. Quantitation: along with requirements of sensitivity, quantitation also requires linearity, accuracy (how close the measured values are to the true values) and precision (how reproducible are those measurements). Investimento: Q-TOF > QQQ ≈ TOF ≈ Ion Trap > Single quad

Il nuovo QTOF Agilent 6520 QTOF Sensibilità, velocità di scansione, accuratezza di massa che ridefiniscono la categoria dei QTOF Più informazioni dai campioni più complessi Sensibilità X10 (Attomoli) Ampio intervallo dinamico (5 decadi) Elevata velocità di scansione (20/sec) e fino a 18,000 di risoluzione Identificazione semplice di composti incogniti Accuratezza di massa X2 (<2 ppm in MS e <5 ppm in MS/MS) Autotune semplice ed efficace Autotune saves time and enhances performance for all operators and samples

Benchmarking – Sensibilità per piccole molecole 200 pg MDMA (3,4-methylenedioxymethamphetamine) 1.61 0.68 400 5.0e5 4.5e5 350 Agilent QTOF 4.0e5 300 Altri QTOF 3.5e5 250 3.0e5 S/N = 230 S/N = 10 2.5e5 200 2.0e5 200pg MDMA, Q1-TTI, CE=0eV, 194.04 (0.1wide) EIC 150 1.5e5 100 1.0e5 50 5.0e4 ! ! 0.2 0.6 1.0 1.4 1.8 0.5 1.0 1.5 2.0 2.5 3.0 Time, min Time, min

Un paragone – Sensibilità su un peptide in MS/MS Infusione ESI, Glu-Fib, MS/MS dell’m/z 876 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 m/z, amu 1800 2000 2200 2400 2600 2800 3000 3200 3400 3600 3800 4000 4200 4400 4600 4800 5000 5200 5400 5600 5800 6000 6200 6400 72.0791 684.3291 187.0662 813.3683 333.1794 480.2426 175.1143 240.1288 120.0774 1056.4458 942.4062 230.0715 1285.5122 1171.4684 87.0529 627.3093 497.1879 102.0522 337.1420 268.1223 924.3967 740.2681 1039.4248 355.1508 612.2114 515.1935 316.1450 159.0733 1268.4917 1570.6306 1154.4547 785.8255 1296.4777 853.2747 1022.3982 409.1285 55.0531 303.2225 482.2451 667.2974 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 m/z, amu 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 0.55 0.60 0.65 0.70 0.75 0.80 0.85 0.90 0.95 1.00 187.06339 333.19855 268.11402 175.12511 480.25381 684.35544 246.16095 785.83143 231.12109 Agilent QTOF Altro QTOF 10-20X sopra il LOD LOD Key message- Agilent QTOF showed strong spectrum of Glu-fib peptide at a level 10 to 20X greater than the other QTOF. Note that the Agilent QTOF clearly sees ions to 1570 m/z that are not detected on the other QTOF. Agilent vs. ABI - 100 fMole/uL Glu-Fib @ 876 m/z (+2) done by infusion as is customary for competitors specification.

Metaboliti: urina maschile/femminile. Identificazione picco a 28 Metaboliti: urina maschile/femminile Identificazione picco a 28.87 minuti

Formula bruta C24H30O6 Error –0.9 ppm Estriol triacetate Ioni pseudomolecolari messi in relazione tra loro

Tabella comparativa degli strumenti QA/QC, verifica e purificazione prodotti Qualitativa Sensibilità SIM (campione semplice) Sensibilità in SIM (campione complesso) Sensibilità in SCAN (campione semplice) Sensibilità in SCAN (campione complesso) Quadrupolo 6 10 7 5 TOF 8 n/a (7) Ion Trap 9 n/a (8) Triplo Quadrupolo Q-TOF n/a (6) Quantitativa di composti target Può fare tutto... Il primo passo dopo il DAD Metabolomica, farmacocinetica, tossicologico Triplo/TOF Screening/Profiling Tossicologico, metabolomica, sintesi, proteomica Note: this table is for internal distribution only. The intent of this table is to assist the salesperson in communicating the relative positioning of instrumentation in the LCMS portfolio. The customers we can best influence using this information are those who are new to LCMS. We want to be especially careful to not offend those experienced customers who have their own opinions. Furthermore, we don’t want the presentation to end up in the hands of our competitors, lest they try to use it against us. Ease of Use: learning curves are a combination of the user’s background and the level of difficulty in working with a particular instrument. We assume that for those customers who have overcome this learning curve and are comfortable with their day-to-day operation, the ease of use is then a function of how many parameters must be explored and optimized for method development. But ease of use may be a bigger issue for a lab with no LCMS experience than for labs which already have some form of LCMS. Scan speed: how long does it take to acquire a full scan MS spectrum for the single quad and TOF, and full scan MS/MS spectrum for the Ion Trap, QQQ and QTOF? Scan sensitivity: how much material is required to obtain a useful full-scan (not MS/MS) spectrum? Along with scan speed, does the instrument perform MS/MS, which is more sensitive for detection, or only MS, which is less sensitive? Qualitative: this ranking depends on how much you know about what you are looking for. A complete unknown may be better identified with accurate mass (empirical formula); a metabolite may be better identified with MS(n) (structure elucidation plus molecular weight). Sensitivity: signal-to-noise using the instrument in its best mode of operation. For an SQ: SIM; QQQ: SRM or MRM; TOF: full scan MS; Ion Trap: full scan MS/MS; and QTOF: full scan MS/MS. Quantitation: along with requirements of sensitivity, quantitation also requires linearity, accuracy (how close the measured values are to the true values) and precision (how reproducible are those measurements). Tossicologico, proteomica, farmaceutico Strumento universale Investimento: Q-TOF > QQQ ≈ TOF ≈ Ion Trap > Single quad

Il portafoglio completo di Agilent Quadrupolo: per sviluppo metodi, screening e quantitativa a basso costo TOF: screening e identificazione in massa accurata Ion Trap: identificazione e quantitativa tramite MSMS QQQ: analisi quantitativa in tracce su matrici complesse Q-TOF: identificazione, screening e quantitativa in massa accurata

Grazie per l’attenzione... marco_zanotti@agilent.com