Slide 1

Seeing is Believing: The Ultrafast LCMS-2020 is the Fastest MS Detector in the World! Analytical & Measuring Instruments Division, Shimadzu Corporation www.partoz.com Tel: +21 88104115 Parto Zist Beboud Slide 2 LAAN-E-LM003 2 Speed is Power UF scanning Ultrahigh scan speed of 15,000 /sec UF switching High-speed positive/negative polarity switching at 15 msec UF sensitivity High sensitivity even in high-speed analysis LCMS-2020 Seeing is Believing Slide 3 LAAN-E-LM003 3 1. Overview of LC/MS Slide 4 Electrospray ionization LAAN-E-LM003 4 Slide 5 Atmospheric pressure LAAN-E-LM003 5 Slide 6 6 Principle of a Mass Spectrometer (MS) Mass Spectrometer A high voltage is applied to the column eluate, and it is atomized by nitrogen gas. The charged droplets gradually become smaller, and ion evaporation occurs. As the ions pass through the mass separator, they are separated according to mass. In the detector, the quantity of ions is detected as a current value. Positive ion (protonated molecule) Proton added Proton removed Negative ion (deprotonated molecule) Proton (hydrogen ion) Separated in a column Mixed sample injected Introduced into MS in order from component with lowest retention Mobile phase Column eluate Ion focusing unit Mass separator Detector Ion source Atmospheric pressure region Vacuum region Slide 7 LAAN-E-LM003 7 Slide 8 8 LC-MS: System Configuration and Ionization Method This is an extremely soft ionization method, and is suited to the ionization of high- polarity compounds. This method is suited to the ionization of medium- and low-polarity compounds. Solvent delivery pump (gradient) Mobile phase Mixer Autosampler Controller LC detector Autosampler cleaning liquid Column oven Column Degasser Reservoir tray Rotary pump Nitrogen gas generator ESI: Electrospray Ionization APCI: Atmospheric Pressure Chemical Ionization Mass spectrometer Glass capillary Charged droplets are formed. Liquid sample Nitrogen gas High voltage (3 to 5 kV) +: Positive ions are created. -: Negative ions are created. Ion evaporation Liquid sample Heater Nitrogen gas Sample molecule Corona needle Solvent molecule High voltage ( 3 to 5 kV) +: Positive ions are created. -: Negative ions are created. Note: The LC detector may be removed if it is not required. Slide 9 LAAN-E-LM003 9 Means of identifying sample Retention time Column Injector Time SPD-20A/20AV SPD-M20A PDA detector Retention time + Mass information LCMS-2020 The molecular weight of the sample can be ascertained with an MS spectrum. Provides greater selectivity than a UV spectrum. 200300400500nm 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 mAU 417 246 600 200300400500nm 0 25 50 75 100 125 150 mAU 421 240 UV spectra Information Obtained from LC and MS Slide 10 LAAN-E-LM003 10 LC-MS: Analytical Data MS m/z time (1) (2) (3) (4) (5) m/z (5) (4) time (3) m/z 193 (2) m/z 582 (1) TIC Mass Chromatogram Mass Spectrum PDA (Photodiode Array) time (3) 210 nm (2) 580 nm LC Chromatogram nm time (2) (3) (4) (5) int. UV Spectrum nm (5) (4) AU Slide 11 LAAN-E-LM003 11 If peak elution is late: If an impurity coincides with the target component: Mobile phase preparation errors Fluctuations in peak retention times Peak misidentification AB AB If an impurity coincides with the target component: Changes in area value Incorrect quantitation Risks Associated with LC Detectors Slide 12 LAAN-E-LM003 12 The greatest merit in using an MS instrument as an LC detector: In addition to retention times, mass information for each peak can be obtained simply at the same time. m/z 264 m/z 278 m/z 267 m/z 281 The peaks (including those that cannot be separated by time) can be separated using mass information. This reduces the risk of qualitative and quantitative errors. Merits of MS Detectors Mass information is a powerful tool for reducing the risks associated with LC analysis, such as the following: Peak identification (i.e., qualitative) errors Quantitative errors due to the elution of unpredicted impurities Slide 13 LAAN-E-LM003 13 2. Features of the LCMS-2020 Slide 14 LAAN-E-LM003 14 With the introduction of the Prominence Ultrafast LC (UFLC), which offers the ultimate level of speed, superior reproducibility, and comprehensive expandability, ultrafast LC analysis is no utilized in a variety of new fields. 0.03.06.09.012.015.0 min. HPLC 0.00.51.01.52.5 min.2.0 UFLC Shimadzu's Ultrafast LC Lineup Prominence UFLC: Greatly reduces analysis time without sacrificing separation. Prominence UFLCXR:Achieves greater separation while maintaining ultrahigh speed. From HPLC to UFLC, Then to UFLC-MS Slide 15 LAAN-E-LM003 15 A mass spectrometer responding to the performance of UFLC: LCMS-2020 What makes an MS instrument suitable for UFLC? The ability to acquire data at high speed without sacrificing data quality is required! The three things that enable ultrafast analysis: The ability to perform scan measurement at high speed UFscanning The ability to switch between positive and negative ion measurement at high speed UFswitching High sensitivity in high-speed measurement UFsensitivity UFLC-MS Slide 16 LAAN-E-LM003 16 20 points10 points 4 to 5 points Influence of Data Sampling Points on Peak Form 1.0 min. UFLC Data If the number of data points decreases, the sensitivity also decreases. This adversely affects the reproducibility. Influence of Data Sampling Points Slide 17 Increase the scan speed. With conventional instruments, the sensitivity decreases. Decrease the scan speed. It is difficult to handle high- speed analysis. LAAN-E-LM003 17 LC-MS: Scan Speed Scan: Data is acquired in the desired m/z range. m/z 100 1,100 Scan speed (scan cycle) t(s) Scan speed (scan cycle) m/z 100 1,100 t(s) Quadrupole rod With the LCMS-2020, it has become possible to maintain sensitivity when the scan speed is increased. Slide 18 LAAN-E-LM003 18 UFscanning With measurement at 15,000 u/sec, the sharp peaks of UFLC are captured reliably. Samples: Seeing is Believing. Slide 19 LAAN-E-LM003 19 1 2 3 5 4 6 7 8 9 10 12 13 14 15 16 11 With measurement at 15,000 u/sec, the sharp peaks of UFLC are captured reliably. Ultrafast analysis of 16 drugs UFscanning 1: Famotidine 2: Cimetidine 3: Atenolol 4: Lidocaine 5: Atropine 6: Metoprolol 7: Yohimbine 8: Noscapine 9: Bupivacaine 10: Alprenolol 11: Tetracaine 12: Diphenhydramine 13: Erythromycin 14: Dibucaine 15: Isopropylantipyrine 16: Warfarin Seeing is Believing. Slide 20 LAAN-E-LM003 20 LC-MS: Positive/Negative Ion Measurement Simultaneous Measurement of Positive and Negative Ions The ease with which positive/negative ions are created depends greatly on the compound characteristics. With positive/negative polarity switching, both positive and negative ions are measured at the same time. In simultaneous measurement, the number of sampling points is important. Peak that readily becomes negative ions Peak that readily becomes positive ions Ionization in negative ion mode Ionization in positive ion mode Chromatograms for both positive and negative ions obtained in a single analysis. 20 points Number of Sampling Points 10 points 4 to 5 points Slide 21 LAAN-E-LM003 21 UFswitching With 15-msec switching during measurement, the sharp peaks of UFLC are captured reliably. Samples: Polarity switching time: Positive ion measurement SIM2CH Negative ion measurement SIM 2CH Positive ion measurement SIM 2CH Slide 22 LAAN-E-LM003 22 UFscanning + UFswitching With measurement at 15,000 u/sec and switching at 15 msec, the sharp peaks of UFLC are captured reliably. Polarity switching time: Positive ion measurement 15,000 u/sec Negative ion measurement 15,000 u/sec Positive ion measurement 15,000 u/sec Mass Spectra of Dymuron Mass Spectra of Carpropamid Mass Spectra of Bentazone Slide 23 LAAN-E-LM003 23 Compounds That Give Both Positive and Negative Ions: Catechins Negative ions 1 2 3 4 5 6 7 8 10 9 1 2 3 4 56 7 8 9 Positive ions m/z 307 m/z 291 m/z 459 m/z 443 m/z 473 m/z 305 m/z 289 m/z 457 m/z 441 m/z 471 Example of the Analysis of Catechins in Tea (Filtered Sample, 2 L) 1: (-)-gallocatechin, 2: (-)-epigallocatechin, 3: (+)-catechin, 4: (-)-epicatechin, 5: (-)-epigallocatechin gallate, 6: (-)-gallocatechin gallate, 7: (-)-epicatechin gallate, 8: (-)-catechin gallate 9, 10: methylated catechins (-)-epicatechin High-Speed Positive/Negative Switching Analysis of Catechins Seeing is Believing. Slide 24 LAAN-E-LM003 24 LCMS-2020 Measurement time + voltage switching time in each mode: 150 msec Measurement time + voltage switching time in each mode: 700 msec Glycyrrhizin Positive Negative Positive Negative High-Speed Positive/Negative Switching Analysis of Glycyrrhizin With measurement at 15,000 u/sec and switching at 15 msec, the sharp peaks of UFLC are captured reliably. Seeing is Believing. Slide 25 LAAN-E-LM003 25 Configuration of the LCMS-2020 Ionization probe Bottle for standard sample Dual inlet turbomolecular pump Mass spectrometric detector Ion optical system 350 mm 726 mm 553 mm Slide 26 LAAN-E-LM003 26 High Sensitivity Even in High-Speed Analysis Qarray Skimmer Octopole Entrance lens To quadrupole rod From desolvation line UFsensitivity Slide 27 LAAN-E-LM003 27 UFsensitivity High sensitivity even in high-speed analysis Reserpine, 1 pg: S/N > 150 (RMS) SIM chromatogram of reserpine (1 pg) 10.015.020.025.030.035.040.045.0 8.25 8.50 8.75 9.00 9.25 (x1,000) 609.30 (1.00) Seeing is Believing. UFsensitivity Slide 28 LAAN-E-LM003 28 LC-MS: Ion Source CID 3 4 1 2 Normal CID Normal CID Normal CID 720 576 158 750 158 592 560 158 718 558 158 716 Ultrafast Analysis of Erythromycin Impurities Set quite a high lens voltage, and produce fragment ions. Slide 29 LAAN-E-LM003 29 A sample was added to blood plasma, acetonitrile was added, centrifugal deproteinization was performed, and the sample was continuously injected in quantities of 1 L. A reproducibility of 2.26% was attained over 10 days, indicating superior durability as well as reproducibility. Ion Source After Continuous Analysis Plasma Sample Injection Number Peak Area of Nortriptyline %RSD: 2.26 Internal standard Analysis time: 6 min 2,500 injections, 10 days Long-Term Stability Features Supporting the Three UFs Slide 30 LAAN-E-LM003 30 Easy Maintenance Simple capillary replacement Large source window Sample bottle for auto-tuning The desolvation line can be replaced without stopping the vacuum. Features Supporting the Three UFs The ionization probe can be attached and detached with a single operation. Slide 31 LAAN-E-LM003 31 A Variety of Ionization Options Molecular weight 10,000 1,000 100 No polarity Medium polarity High polarity ESI DUIS APCI Features Supporting the Three UFs ESI probe Introduction of sample Corona needle Slide 32 LAAN-E-LM003 32 A Variety of Ionization Options Features Supporting the Three UFs MS Chromatograms MS Spectra Obtained with DUIS Measurement Thiamine Riboflavin Calciferol 3 Types of Water-Soluble/Lipid-Soluble Vitamins (Mixed Sample) 1. Thiamine:m/z 265:Cations, water-soluble vitamin created by dissociation 2. Riboflavin:m/z 377:Protonated molecules, water-soluble vitamin 3. Calciferol:m/z 397:Protonated molecules, lipid-soluble vitamin Slide 33 LAAN-E-LM003 33 LCMSsolution ver.5 The popular aspects of LCsolution and LCMSsolution Ver. 3 have been retained, and an easier-to-use interface has been added. LCMSsolution Ver. 5 Slide 34 LAAN-E-LM003 34 Comparison of PDA and MS chromatograms Comparison of chromatograms in different data files Switching between parallel display and overlap display New Functions of Data Browser Slide 35 LAAN-E-LM003 35 Searches for the optimum lens voltage for each compound Sets optimum values in method files Automatic Optimization of MS Parameters Slide 36 LAAN-E-LM003 36 3. Electrospray VS MALDY Slide 37 LAAN-E-LM003 37 Slide 38 LAAN-E-LM003 38 Slide 39 LAAN-E-LM003 39 Slide 40 LAAN-E-LM003 40 Slide 41 LAAN-E-LM003 41 Slide 42 LAAN-E-LM003 42 Slide 43 LAAN-E-LM003 43 4. LCMS-2020: Influence of Mobile Phase Solvent Slide 44 LAAN-E-LM003 44 Xanthine Derivatives 0.1% Aqueous Formic Acid/MeOH = 80/20 Shim-pack VP-ODS 2 50mm, 5m 0.2mL/min Y = (81193.2)X + (1.72242e+006) R2 = 0.99952 Y = (37825.1)X + (880292) R2 = 0.99920 Y = (37263.6)X + (612820) R2 = 0.99954 Caffeine Theophylline Theobromine Slide 45 45 LAAN-E-LM003 Xanthine Derivatives XR-ODS 2 75mm, 2.2 m 0.4 mL/min Prioritizing maintenance of the separated state Theobromine in Green Tea Elution Time 9 min 2.2 min Reduced by 6.8 min Solvent Volume 1.2 mL 0.88 mL 51% OFF 0.1% Aqueous Formic Acid/MeOH = 80/20 Area Concentration Theobromine Slide 46 LAAN-E-LM003 46 Xanthine Derivatives XR-ODS 2 50 mm, 2.2 m 0.4m L/min Specifying the column size Theobromine in Green Tea Elution Time 9 min 1.6 min Reduced by 7.4 min Solvent Volume 1.8 mL 0.64 mL 64% OFF 0.1% Aqueous Formic Acid/MeOH = 80/20 Theobromine Area Concentration Slide 47 LAAN-E-LM003 47 Catechins: ESI(-) 0.5% Aqueous Formic Acid/ACN 2% (0 min) - 25% (3 min) - 2% (3.01-5 min) Gradient XR-ODS 2 50 mm, 2.2 m 0.5 mL/min 0.5% Aqueous Formic Acid/ACN/THF MAX 19.2 MPa 1: Gallic acid 2: (-)-gallocatechin, 3: (-)-epigallocatechin, 4: (+)-catechin, 5: (-)-epicatechin, 6: (-)-epigallocatechin gallate, 7: (-)-gallocatechin gallate, 8: (-)-epicatechin gallate, 9: (-)-catechin gallate 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 *Adding THF improves epimer separation. *Peaks 5 and 6 are also separated well. 2% (0 min) - 25% (3 min) - 2% (3.01-5 min) Gradient Slide 48 LAAN-E-LM003 48 Catechins: ESI(-) 0.5% Aqueous Formic Acid/MeOH MAX 27.5 MPa 1 2 3 4 5 6 7 8 9 0.5% Aqueous Formic Acid/ACN Same analysis time Mobile phase B Acetonitrile Methanol *Epimer separation is better. *Sensitivity is also good. *There is insufficient separation between peaks 3 and 4 although this is not a problem with LCMS. MAX 19.2 MPa 1 2 3 4 5 6 7 8 9 XR-ODS 2 50 mm, 2.2 m 0.5 mL/min 2% (0 min) - 25% (3 min) - 2% (3.01-5 min) Gradient 3% (0 min) - 50% (3 min) - 3% (3.01-5 min) Gradient Slide 49 LAAN-E-LM003 49 Carbamate Pesticides [M+H] + : m/z 207[M+NH 4 ] + : m/z 240[M+H] + : m/z 237[M+H] + : m/z 163[M+H] + : m/z 242 [M+H-H 2 O] + : m/z 220 [M+H] + : m/z 258[M+NH 4 ] + : m/z 208[M+H] + : m/z 224[M+H] + : m/z 222 [M+H] + : m/z 202[M+H] + : m/z 355[M+H] + : m/z 226[M+H] + : m/z 180 (1) Aldicarb Sulfoxide(2) Aldicarb Sulfone(3) Oxamyl(4) Methomyl(5) Methiocarb Sulfoxide (6) 3-OH Carbofuran(7) Methiocarb Sulfone(8) Aldicarb(9) Bendiocarb(10) Carbofuran (11) Carbaryl(12) Thiodicarb(13) Ethiofencarb(14) XMC [M+H] + : m/z 239 [M+H] + : m/z 194 [M+H] + : m/z 208[M+H] + : m/z 226[M+H] + : m/z 411 (15) Pirimicarb (16) Isoprocarb(17) Trimethacarb(18) Fenobcarb(19) Methiocarb(20) Benfuracarb Slide 50 LAAN-E-LM003 50 Carbamate Pesticides 10-mmol/L Aqueous Ammonium Acetate/ACN 5% (0 min) - 95% (20-25 min) - 5% (25.01-35 min) Gradient 10-mmol/L Aqueous Ammonium Acetate/MeOH VP-ODS 2 150 mm, 5 m, 0.2 mL/min 1 2 3 4 5 6 7 8 9 10 11 12 13 20 16 17 19 18 14 15 1 2 3 4 5 6 7 8 9 10 11 12 13 20 17 19 18 14 15 16 *It is difficult to separate peaks 16 and 17 with acetonitrile, so use of methanol is recommended for this analysis. *Sensitivity is also better with methanol. 5% (0 min) - 95% (20-25 min) - 5% (25.01-35 min) Gradient Slide 51 LAAN-E-LM003 51 Carbamate Pesticides 10-mmol/L Aqueous Ammonium Acetate/MeOH 5% (0 min) - 95% (3-4 min) - 5% (4.01-8 min) Gradient XR-ODS 2 50 mm, 2.2 m, 0.4 mL/min MAX 25.5 MPa Analysis Time 40 min 8 min Reduced by 32 min Solvent (Methanol) Volume 3.05 mL 1.06 mL 65% OFF 1 2 3 4 5 6 7 8 9 10 11 13 20 16 17 19 18 14 15 12 Slide 52 LAAN-E-LM003 52 14 Drugs [M+H] + : m/z 338[M+H] + : m/z 253[M+H] + : m/z 267[M+H] + : m/z 315 [M+H] + : m/z 237[M+H] + : m/z 235[M+H] + : m/z 355[M+H] + : m/z 250[M+H] + : m/z 280 [M+H] + : m/z 281[M+H] + : m/z 278[M-H] : m/z 329[M+H] + : m/z 231[M+H] + : m/z 309 [M-H] : m/z 307 (1) famotidine (2) cimetidine(3) atenolol(4) ranitidine (5) procaine(6) lidocaine(7) yohimbine(8) alprenolol(9) doxepin (10) imipramine(11) amitriptyline(12) furosemide(13) isopropylantipyrine(14) warfarin Slide 53 LAAN-E-LM003 53 14 Drugs 0.1% Aqueous Formic Acid/ACN 1 2 3 4 5 6 7 8 9 10 11 12 13 14 XR-ODS 2 50 mm, 2.2 m 0.4 mL/min 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Analysis Time 6.5 min 4 min Reduced by 1.5 min Solvent Volume 1.06 mL 0.36 mL 66% OFF *Analysis can be performed in the same way even if the gradient conditions are changed; as a result, the analysis time can be reduced. 5% (0 min) - 80% (3-4 min) - 5% (4.01-8 min) Gradient 5% (0 min) - 80% (1-1.5 min) - 5% (1.51-5 min) Gradient Slide 54 LAAN-E-LM003 54 14 Drugs 0.1% Aqueous Formic Acid/MeOH 0.1% Aqueous Formic Acid/ACN 1 2 3 4 5 6 7 8 9 10 11 12 13 14 6 7 8 9 10 11 12 13 14 1 2 3 4 5 Same analysis time Mobile phase B Acetonitrile Methanol *It is difficult to separate peaks 1 to 5. XR-ODS 2 50 mm, 2.2 m 0.4 mL/min 5% (0 min) - 80% (3-4 min) - 5% (4.01-8 min) Gradient 5% (0 min) - 85% (3-4 min) - 5% (4.01-8 min) Gradient Slide 55 LAAN-E-LM003 55 14 Drugs 1 2 3 4 5 6 7 8 9 10 11 12 13 14 10-mmol/L Aqueous Ammonium Acetate/MeOH 6 7 8 9 10 11 12 13 14 1 2 3 4 5 Same analysis time Mobile phase B Acetonitrile Methanol Mobile phase A 0.1% aqueous formic acid 10-mmol/L aqueous ammonium acetate 0.1% Aqueous Formic Acid/ACN *Peaks 1 to 5 are separated well. *Sensitivity is at least as good. *The elution order changes but, with LC/MS, identification is simple. XR-ODS 2 50 mm, 2.2 m 0.4 mL/min 5% (0 min) - 85% (3-4 min) - 5% (4.01-8 min) Gradient 5% (0 min) - 80% (3-4 min) - 5% (4.01-8 min) Gradient Slide 56 LAAN-E-LM003 56 14 Drugs 10-mmol/L Aqueous Ammonium Acetate/ACN 10-mmol/L Aqueous Ammonium Acetate/MeOH 6 7 8 9 10 11 12 13 14 1 2 3 4 5 1 2 3 4 5 6 7 8 9 10 11 12 13 14 *The elution order changes but, with LC/MS, identification is simple. XR-ODS 2 50 mm, 2.2 m 0.4 mL/min 5% (0 min) - 85% (1-1.5 min) - 5% (1.51-5 min) Gradient 5% (0 min) - 65% (1-1.5 min) - 5% (1.51-5 min) Gradient Slide 57 LAAN-E-LM003 57 A mass spectrometer responding to the performance of UFLC: LCMS-2020 With the LCMS-2020, the peaks output from the Prominence UFLC at high speed can be captured. High sensitivity can be attained even in high-speed measurement. It is easy to change from acetonitrile to methanol. - Sensitivity - Pressure - Changes in separation patterns Summary Slide 58 LAAN-E-LM003 58 Technical Reports on Ways of Saving Solvent No. 24 Reduced Solvent Consumption and Operational Costs with Ultra High Speed Analysis No. 25 Reduced Solvent Consumption and Operational Costs with Existing LC Systems Related Technical Reports