Interfacing UPC 2 to MS

Interfacing UPC2 to MS

Andrea Perissi Specialista Applicativo, Waters Italia

 Introduction  Traditional approaches to interfacing SFC to MS  Ionization Mode  Approach used for UPC2  Examples

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

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

 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

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

 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

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

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

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

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

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

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 Minutes

18:0/18:0

High energy

18:1/18:1

18:2/18:2

Low energy

18:3/18:3

Free fatty acids ESI- (FFA)

Triacylglycerols (TAG)

ESI+ Chosteryl esters (CE)

Mixture of FFA, TAG, and CE

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

 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.

 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

 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

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)

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.

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.

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

 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

 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

 ACQUITY UPC2 system with PDA  Flow rate: 1 mL/min  Co-solvent (mobile phase B): acetonitrile

 Column: ACQUITY UPLC HSS C18 (3.0 x 100 mm, 1.7 µm)  Backpressure: 2500 psi  Temperature: 30 C  Sample diluent: MTBE  Injection volume: 1 µL  PDA scan range: 210-600 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)

0.40 0.38 0.36 0.34

0.32 K1 E 0.30

0.28 Beta carotene 0.26 A Acetate 0.24 0.22 D2 0.20 AU 0.18 A Palmitate 0.16 Lycopene

0.14 E Acetate 0.12

0.10 K2 0.08 0.06 0.04 0.02 0.00

0.00 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 3.00 3.20 3.40 3.60 3.80 4.00 Minutes

Retention Analyte λ (nm) Time (min)

(1) Vit A Acetate 3.14 345

(2) Vit E Acetate 3.76 263

(3) Vit K1 3.88 263

(4) Vit A 4.34 345 Vitamin A Palmitate Normal phase (5) Vit E 4.82 263 12 minutes Tocopherol 2 9 10 (6) Lycopene 4.99 456 Vitamin D3 3 (7) Vit E Normal phase 5.09 263 5 Succinate 20 minutes (8) β-carotene 5.12 456 7,8 Vitamin E (9) Vit D3 5.76 263 Normal phase (10) Lutein 7.21 456 30 minutes 1 AU β-carotene 6 Normal phase 4 10 minutes

Lycopene Normal phase 10 minutes

Vitamin K1 Reversed-phase 0.00 2.00 4.00 6.00 8.00 10.00 12 minutes Minutes

Lutein Reversed-phase Simultaneous Analysis of Fat-soluble Vitamins 10 minutes and Carotenoids in 10 minutes

 Simultaneous separation of 9 fat-soluble vitamins and carotenoids in 4 min using a single-injection UPC2 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

 UPC2 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

 Acquity UPC2 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