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Interfacing UPC 2 to MS Interfacing UPC2 to MS Andrea Perissi Specialista Applicativo, Waters Italia ©2012 Waters Corporation 1 Agenda Introduction Traditional approaches to interfacing SFC to MS Ionization Mode Approach used for UPC2 Examples ©2012 Waters Corporation 2 Separation Technology Overview Separation achieved by a temperature gradient •High efficiency [N] GC • Virtually no limitation on column length •Limited selectivity [α] • Limited stationary phase options Gas Chromatography Separation achieved by a solvent gradient •High efficiency [N] LC • Limited to pressure drop across column • Moderate selectivity [α] • Different modes: reversed-phase, normal-phase, SEC, IEX, affinity, ion pair, HILIC, GPC…etc. Liquid Chromatography Separation achieved by density/solvent gradient •High efficiency [N] CC • Very low viscosity enables longer columns and smaller particles •High selectivity [α] • Wide variety of stationary phase and mobile phase co-solvent and modifier options Convergence Chromatography ©2012 Waters Corporation 3 The Advent of Convergence Chromatography UltraPerformance SFC Convergence Chromatography UltraPerformance Convergence Chromatography is the result of significant technological advancements in Supercritical Fluid Convergence Chromatography Chromatography instrumentation and chemistry design while providing exceptional increases in available selectivity ©2012 Waters Corporation Data courtesy of Davy Guillarme, Jean-Luc Veuthey LCAP, University of Geneva, Switzerland 4 What is UPC2? Based on Acquity UPLC TM holistic design Based on SFC but uses sub-2µm particle to increase chromatographic performance such as – Speed of separation – Peak capacity – Complements to MS due to its low solvent load Uses CO2 as a major solvent and co-solvents such as methanol to vary the mobile phase strength ©2012 Waters Corporation 5 ACQUITY UPC2 System Over 20 patent applications!! ACQUITY UPC2 Detection •Photodiode array •Evaporative light scattering •Mass spectrometry ACQUITY UPC2 Column Manager Select from up to 6 columns for method screening ACQUITY UPC2 Convergence Manager •Manages CO2 inlet •Auxiliary inject valve •Active back pressure regulator ACQUITY UPC2 Sample Manager Fixed-loop injector Perform Partial Loop Injections ACQUITY UPC2 Binary Solvent Manager Pumps liquid CO2 and desired co-solvent (select from up to 4 co-solvents) ©2012 Waters Corporation 6 Acquity UPC 2 Approach We use post column pre-convergence manager split. (fixed leak) — Below 5% co-solvent requires a pre-split makeup pump. — Typically results in about 40uL/ min into the source with the makeup pump at 450uL/ min Utilize 30 in of 50u peeksil to source from split — Normally an issue with LC, we don’t have a laminar flow issue with longer rubing to MS Can make use of either Esi or APcI with ESCi source ©2012 Waters Corporation 7 ACQUITY UPC2/MS Splitter Configuration ©2012 Waters Corporation 8 Gradient Mix Overlay of UV and MS with Waters Splitter ©2012 Waters Corporation 9 Compatibility with Mass Spectrometry Single Quadrupoles Tandem Quadrupoles SQD SQD2 TQD XEVO TQD Time of Flight (ToF) XEVO TQ-MS XEVO TQ-S XEVO G2-S TOF ACQUITY UPC2 System ©2012 Waters Corporation 10 Addressing Selectivity: Convergence Chromatography Solvent Stationary Phase Pentane, Hexane, Heptane Silica / BEH Xylene Toluene 2-ethylpyridine Diethyl ether Dichloromethane Cyano Chloroform Convergence Aminopropyl Acetone Chromatography Dioxane Selectivity Space Diol THF Unlimited solvent MTBE and stationary Amide Ethyl acetate phase selectivity DMSO PFP Acetonitrile Isopropanol Phenyl Ethanol C < C Methanol 18 8 ©2012 Waters Corporation 11 ACQUITY UPC2 System: Expanding the Selectivity Space ACQUITY UPC2 CSH Fluoro-Phenyl 1.7 µm : CSH PFP 0.048 B C 0.036 API 0.024 D AU A (1,2) E F G H 0.012 0.000 : HSS C18 SB 2 0.048 ACQUITY UPC HSS C18 SB 1.7 µm 0.036 C B 0.024 A D API AU G F 0.012 E H 0.000 : BEH HILIC 0.048 ACQUITY UPC2 Hybrid 1.7 µm 0.036 B API C D 0.024 G AU A H 0.012 E F* 0.000 : 2-EP 2 0.048 ACQUITY UPC Hybrid 2-EP 1.7 µm B C 0.036 D E API 0.024 G AU A H 0.012 F 0.000 0.00 0.60 1.20 1.80 2.40 3.00 3.60 4.20 4.80 5.40 6.00 Minutes ©2012 Waters Corporation 12 UPC2 Applications by Market Pharmaceutical Food/Env Chemical Materials Clinical Chiral drugs Transfer from USP methods Fat-soluble vitamins Lipid analysis Achiral impurity analysis Chiral pesticides Explosives OLEDs Azo dyes Non-ionic surfactants Polymer additives Drugs of abuse Vitamin D metabolites Carotenoids Positional isomers Steroids Steroids Extractables and leachables Reaction monitoring Library screening DMPK/Bioanalysis Natural products Metabolomics Glycerides PAH Lubricants ©2012 Waters Corporation 13 Lipids Analysis ©2012 Waters Corporation 14 Separation of Different Lipid Classes in Mouse Heart Extract CER Ceramides SM Sphingomyelin PG Phosphatidylglycerol PE Phosphatidylethanolamine 8x107 PC ES+ SIR Overlays PC Phosphatidylcholine LPC Lyso-Phosphatidylcholine LPE Lyso- Phosphatidylethanolamine 6x107 4x107 Intensity 2x107 LPC PE SM CER PG LPE 0 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 Minutes ©2012 Waters Corporation 15 MSE using UPC2/oa-QTof 18:0/18:0 High energy 18:1/18:1 18:2/18:2 Low energy 18:3/18:3 ACQUITY UPC2 BEH *Performed on a Synapt G2 ©2012 Waters Corporation 16 Separation of Neutral Lipids Free fatty acids ESI- (FFA) Triacylglycerols (TAG) ESI+ Chosteryl esters (CE) Mixture of FFA, TAG, and CE ©2012 Waters Corporation 17 Free Fatty Acid Analysis FFA Mix 1 FFA Method 0.5ul inj 15CV0 17:14:13 C22 03-Aug-2012 TAGs_08022012_026 1: TOF MS ES- 100 1.72 BPI C20339.3196 1.71e5 1.52 C18311.2894 C24 Method Conditions: 1.35 2 1.93 Instrument: ACQUITY UPC C16283.2587 367.3502 2 Column: ACQUITY UPC HSS C18 SB 1.18 Co-solvent: MeOH w/ 2g/L Ammonium C14255.2276 Formate Gradient: 1 to 10% over 5 minutes* 1.05 227.1970 Flow: 2.5 mL/min Temperature: 600C CCM Pressure: 1885 % Injection Vol: 0.5uL C12 Make-up flow: 0.2mL/min of 0.1% formic acid 0.91 199.1659 Sample Concentration: 0.25 mg/mL FFA: C8 to C24 C10 0.79 171.1348 C8 0 Time 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 ©2012 Waters Corporation 18 The UPC² Advantages Rapid screening of lipids by class – Method selective with other biological extracts. (i.e mouse heart extract) Separation of lipids based on acyl chain length and double bond position ACQUITY UPC² compared to other techniques: – Vs. HILIC, UPC² benefits are o Utilizing less organic solvent o Less equilibration than HILIC approach, more user friendly – Vs. Reversed Phase – LC, UPC² benefits are o Better separations of interclass moieties. ©2012 Waters Corporation 19 Conversion of USP GC Method to Convergence Chromatography: Analysis of Atropa Bella-donna ©2012 Waters Corporation 20 Background Atropa belladonna extract is primarily composed of the tropane alkaloids, scopolamine and hyoscyamine (atropine) The current United States Pharmacopoeial Convention (USP) method specifies a gas chromatography (GC) method High temperature required for GC analyses can lead to dehydration and conversion of scopolamine and atropine to their apo-forms. — Issues are often resolved by derivatization ©2012 Waters Corporation 21 Conditions ACQUITY UPC² with PDA and SQD detection Modifier (B): 0.2% NH4OH (28-30%) in 98/2 MeOH/H2O Column: ACQUITY UPC² BEH 3.0 x 100 mm, 1.7 µm Gradient:10% to 30% B in 4.5 min, 30% to 40% in 0.5 min, 40% to 5% in 0.5 min Flow at 2 mL/min ABPR: 2000psi Temp: 50°C Wavelength: 220 nm, compensated (410- 480 nm) Column temperature: 50 °C Injection volume: 1 µL Conditioning parameters: 1 mL/min, 100% Modifier, 2.5 h ©2012 Waters Corporation 22 System Suitability Criteria Parameter USP Criteria UPC² Analysis (GC Method) Relative standard deviation of <2.0 < 1.6 six to ten injections for the ratio RA/RH Resolution, R, between αH and >3.0 > 3.9 αA Tailing factor measured at 5% of <2.0 < 1.5 the peak height of aA Linear dynamic range for 3x 40x scopolamine and atropine (orders of magnitude) ©2012 Waters Corporation 23 Reproducibility 0.030 Atropine 0.025 Scopolamine 0.020 0.015 AU Homatropine 0.010 (IS) 0.005 0.000 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 Minutes Overlay of eight replicate UPC² separations of Atropa belladonna extract standards, scopolamine, and atropine (200 µg/mL); homatropine internal standard (50 µg/mL) UV at 220 nm compensated wavelength. ©2012 Waters Corporation 24 Linearity Scopolamine Atropine R2 0.9995 0.9998 S/N LOQ at 0.001% 27.5 17.4 0.060 Scopolamine 0.050 0.040 Atropine 0.030 AU Homatropine 0.020 (IS) 0.010 0.000 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 Minutes Overlay of linearity calibration standards from 10 to 400 µg/mL. ©2012 Waters Corporation 25 Chromatograms of Commercial Extract Preparation Atropine Analyte m/z 1.2x108 MS SIR 1.0x108 Scopolamine 304.4 Homatropine 8.0x107 Homatropine 276.3 (IS) 6.0x107 Atropine 290.4 Intensity 7 Scopine 154.4 4.0x10 Scopine Scopolamine 2.0x107 0.0 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 0.12 Minutes 0.10 UV @ 220 nm 0.08 0.06 AU Homatropine 0.04 Scopine (IS) 0.02 Atropine 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 Minutes ©2012 Waters Corporation 26 Benefits of UPC2 Provides alternative to gas chromatography for heat labile compounds.
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