Andrea Perissi Specialista Applicativo, Waters Italia Waters Corporation 1

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1 Interfacing UPC 2 to MS Andrea Perissi Specialista Applicativo, Waters Italia 2012 Waters Corporation 1

2 Agenda Introduction Traditional approaches to interfacing SFC to MS Ionization Mode Approach used for UPC 2 Examples 2012 Waters Corporation 2

3 Separation Technology Overview Separation achieved by a temperature gradient Gas Chromatography GC High efficiency [N] Virtually no limitation on column length Limited selectivity [α] Limited stationary phase options Liquid Chromatography LC Separation achieved by a solvent gradient High efficiency [N] Limited to pressure drop across column Moderate selectivity [α] Different modes: reversed-phase, normal-phase, SEC, IEX, affinity, ion pair, HILIC, GPC etc. Separation achieved by density/solvent gradient Convergence Chromatography CC High efficiency [N] 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 2012 Waters Corporation 3

4 The Advent of Convergence Chromatography SFC UltraPerformance Convergence Chromatography Convergence Chromatography UltraPerformance Convergence Chromatography is the result of significant technological advancements in Supercritical Fluid 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

5 What is UPC 2? 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 CO 2 as a major solvent and co-solvents such as methanol to vary the mobile phase strength 2012 Waters Corporation 5

6 ACQUITY UPC 2 System Over 20 patent applications!! ACQUITY UPC 2 Detection Photodiode array Evaporative light scattering Mass spectrometry ACQUITY UPC 2 Column Manager Select from up to 6 columns for method screening ACQUITY UPC 2 Convergence Manager Manages CO 2 inlet Auxiliary inject valve Active back pressure regulator ACQUITY UPC 2 Sample Manager Fixed-loop injector Perform Partial Loop Injections ACQUITY UPC 2 Binary Solvent Manager Pumps liquid CO 2 and desired co-solvent (select from up to 4 co-solvents) 2012 Waters Corporation 6

7 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

8 ACQUITY UPC 2 /MS Splitter Configuration 2012 Waters Corporation 8

9 Gradient Mix Overlay of UV and MS with Waters Splitter 2012 Waters Corporation 9

10 Compatibility with Mass Spectrometry Single Quadrupoles Tandem Quadrupoles SQD SQD2 Time of Flight (ToF) TQD XEVO TQD XEVO TQ-MS XEVO TQ-S ACQUITY UPC 2 System XEVO G2-S TOF 2012 Waters Corporation 10

11 Addressing Selectivity: Convergence Chromatography Solvent Pentane, Hexane, Heptane Xylene Toluene Diethyl ether Dichloromethane Chloroform Acetone Dioxane THF MTBE Ethyl acetate DMSO Acetonitrile Isopropanol Ethanol Methanol Convergence Chromatography Selectivity Space Unlimited solvent and stationary phase selectivity Stationary Phase Silica / BEH 2-ethylpyridine Cyano Aminopropyl Diol Amide PFP Phenyl C 18 < C Waters Corporation 11

12 ACQUITY UPC 2 System: Expanding the Selectivity Space : CSH PFP ACQUITY UPC 2 CSH Fluoro-Phenyl 1.7 µm B C API A U A D (1,2) E F G H : HSS C18 SB ACQUITY UPC 2 HSS C 18 SB 1.7 µm A U A B C D F G E H API A U : BEH HILIC ACQUITY UPC 2 Hybrid 1.7 µm A B C D G E API F* H A U : 2-EP ACQUITY UPC 2 Hybrid 2-EP 1.7 µm A B C G D E API H F Minutes 2012 Waters Corporation 12

13 UPC 2 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 Extractables and leachables Reaction monitoring Library screening DMPK/Bioanalysis Natural products Metabolomics Glycerides PAH Lubricants Steroids 2012 Waters Corporation 13

14 Lipids Analysis 2012 Waters Corporation 14

15 Separation of Different Lipid Classes in Mouse Heart Extract CER Ceramides SM Sphingomyelin 8x10 7 PG PE PC Phosphatidylglycerol Phosphatidylethanolamine Phosphatidylcholine PC ES+ SIR Overlays LPC Lyso-Phosphatidylcholine LPE Lyso- Phosphatidylethanolamine 6x10 7 Intensity 4x10 7 2x10 7 PE LPC SM CER PG LPE Minutes 2012 Waters Corporation 15

16 MS E using UPC 2 /oa-qtof 18:0/18:0 High energy 18:1/18:1 18:2/18:2 Low energy 18:3/18:3 ACQUITY UPC 2 BEH *Performed on a Synapt G Waters Corporation 16

17 Separation of Neutral Lipids ESI- Free fatty acids (FFA) Triacylglycerols (TAG) ESI+ Chosteryl esters (CE) Mixture of FFA, TAG, and CE 2012 Waters Corporation 17

18 Free Fatty Acid Analysis FFA Mix 1 FFA Method 0.5ul inj 15CV0 17:14:13 TAGs_ _ % C C C C C22 C Aug : TOF MS ES- BPI 1.71e5 Method Conditions: Instrument: ACQUITY UPC 2 Column: ACQUITY UPC 2 HSS C 18 SB Co-solvent: MeOH w/ 2g/L Ammonium Formate Gradient: 1 to 10% over 5 minutes* Flow: 2.5 ml/min Temperature: 60 0 C CCM Pressure: 1885 Injection Vol: 0.5uL C10 C Make-up flow: 0.2mL/min of 0.1% formic acid Sample Concentration: 0.25 mg/ml FFA: C8 to C24 C Time 2012 Waters Corporation 18

19 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 Waters Corporation 19

20 Conversion of USP GC Method to Convergence Chromatography: Analysis of Atropa Bella-donna 2012 Waters Corporation 20

21 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

22 Conditions ACQUITY UPC² with PDA and SQD detection Modifier (B): 0.2% NH 4 OH (28-30%) in 98/2 MeOH/H 2 O 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 ( nm) Column temperature: 50 C Injection volume: 1 µl Conditioning parameters: 1 ml/min, 100% Modifier, 2.5 h 2012 Waters Corporation 22

23 System Suitability Criteria Parameter USP Criteria (GC Method) UPC² Analysis Relative standard deviation of six to ten injections for the ratio R A /R H <2.0 < 1.6 Resolution, R, between αh and αa >3.0 > 3.9 Tailing factor measured at 5% of <2.0 < 1.5 the peak height of a A Linear dynamic range for scopolamine and atropine (orders of magnitude) 3x 40x 2012 Waters Corporation 23

24 Reproducibility Scopolamine Atropine AU Homatropine (IS) 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 Waters Corporation 24

25 Linearity Scopolamine Atropine R S/N LOQ at 0.001% Scopolamine Atropine AU Homatropine (IS) Minutes Overlay of linearity calibration standards from 10 to 400 µg/ml Waters Corporation 25

26 Chromatograms of Commercial Extract Preparation Intensity 1.2x x x x10 7 MS SIR Homatropine (IS) Atropine Analyte m/z Scopolamine Homatropine Atropine x x10 7 Scopolamine Scopine Scopine Minutes 220 nm 0.08 AU Scopine Homatropine (IS) Atropine Minutes 2012 Waters Corporation 26

27 Benefits of UPC 2 Provides alternative to gas chromatography for heat labile compounds. Does not require derivatization Robust analysis is completed in less than five minutes Linear dynamic range from 10 to 400 µg/ml Compatible with mass spectrometry for identification of unknown components 2012 Waters Corporation 27

28 Simultaneous Separation of Fat Soluble Vitamins and Carotenoids 2012 Waters Corporation 28

29 Background Fat-soluble vitamins are involved in complex metabolic reactions related to important biological functions Beta-carotene is a precursor to vitamin A and has 100% vitamin A activity in the body Lycopene is not an essential nutrient for humans, but due to its potential antioxidation benefit, it is also marketed in some dietary supplements along with other ingredients Traditionally analyzed by HPLC (both NP and RP) Extracts can be directly injected onto NP RP gives better resolution, but not compatible with extracts and run time is long Lack of methods for simultaneous analysis of fat-soluble vitamins in premixes, as well as for analyzing carotenoids 2012 Waters Corporation 29

30 Structures 2012 Waters Corporation 30

31 Conditions ACQUITY UPC 2 system with PDA Flow rate: 1 ml/min Co-solvent (mobile phase B): acetonitrile Column: ACQUITY UPLC HSS C 18 (3.0 x 100 mm, 1.7 µm) Backpressure: 2500 psi Temperature: 30 C Sample diluent: MTBE Injection volume: 1 µl PDA scan range: nm Gradient: Hold at 2% B for 2 min, then to 20% B in 0.5 min, hold at 20% B for 1 min, reset (4 min total run time) 2012 Waters Corporation 31

32 UPC 2 Chromatogram A Acetate K1 E Beta carotene AU E Acetate D2 A Palmitate Lycopene K Minutes 2012 Waters Corporation 32

33 Combining Multiple LC Methods Fat-soluble vitamins Analyte Retention Time (min) λ (nm) (1) Vit A Acetate (2) Vit E Acetate (3) Vit K Vitamin A Normal phase 12 minutes Vitamin D3 Normal phase 20 minutes Vitamin E Normal phase 30 minutes β-carotene Normal phase 10 minutes AU , (4) Vit A Palmitate (5) Vit E Tocopherol (6) Lycopene (7) Vit E Succinate (8) β-carotene (9) Vit D (10) Lutein Lycopene Normal phase 10 minutes Vitamin K1 Reversed-phase 12 minutes Lutein Reversed-phase 10 minutes Minutes Simultaneous Analysis of Fat-soluble Vitamins and Carotenoids in 10 minutes 2012 Waters Corporation 33

34 Benefits of UPC 2 Simultaneous separation of 9 fat-soluble vitamins and carotenoids in 4 min using a single-injection UPC 2 method 4-10 times faster than LC Can be used by food/supplement industries for regulatory compliance monitoring where a large number of analyses are required UPC 2 eliminates the need for multiple methods often required for analyzing such mixtures using LC Reduces the number of assays for laboratories routinely performing multiple fat-soluble vitamin analyses 2012 Waters Corporation 34

35 Summary Acquity UPC 2 can be successfully interfaced to all types of MS Multiple ionization modes can be utilized Minimal Bandspread allows use of sub 2u chemistries Sensitivity and Carryover the same or better than LC Alternative to NP interface to MS for chiral separations Makeup-pump allows additives to help promote ionization 2012 Waters Corporation 35

36 Questions? 2012 Waters Corporation 36