from uhplc to hplc-chip/ms, agilent’s ultimate separation ... uhplc to hplc... · from uhplc to...

From UHPLC to HPLC-Chip/MS, Agilent’s Ultimate Separation Tools for LC/MS

Dayin Lin, Ph.D.Dayin Lin, Ph.D.Agilent Technologies, Inc.

Topics

• Agilent LC Overview

Choices of LC for LC/MS• Choices of LC for LC/MS

• Typical Performance Data

• Application Examples• DBS for DMPK

Bi h ti l A l i• Biopharmaceutical Analysis

• Summary

UHPLC to HPLC-Chip/MS2

Agilent’s Comprehensive LC Portfolio

Preparative LC Standard LC UHPLC incl. RRLC Capillary &

Nano LCHigh Sensitivity HPLC-Chip/MS

• Open Platform with >60 Modules

Some Recent Enhancements• New Multi-Method and Method Development System • New and Improved Detector Choices (ELSD and VWD)

• Modular and Scalable

• Enables Stepwise Upgrades

p ( )• HiP Autosampler SL+ with higher precision and reduced

carryover• Lab Advisor for Improved Diagnostics, Maintenance and

ReportingReporting


Agilent 1290 Infinity LC System - Infinitely BetterCovering UHPLC AND HPLC SolutionsgInfinitely more powerful

– with system pressure up to 1200 bar at 2 ml/min and flow rates up to 5 mL/min2 ml/min and flow rates up to 5 mL/min at 800 bar,

Infinitely more sensitive– New diode array detector gives highest

sensitivity in UV detectionInfinitely more flexible

– the new binary pump delivers lowest delay for ultra fast gradientsdelay for ultra-fast gradients

– with seamless method transfer from any UHPLC or HPLC system

– The new autosampler and 1290 Infinity p yFlexible Cube provides ultimate flexibility in sample injection –flow-through or fixed loop


Method Development SystemsApplication-based LC Solutionspp

The most versatile system for analytical method development

Integrated with Automatic method optimization software

available• Automated switching between up to

8 columns • Automated switching between up to

availablefrom

Agilent‘s partners

g p26 solvents to maximize separation selectivity

• Accommodates column lengths up to 300 mm and inner diameters of 2.1 to 4.6 mm

• Systems available from 400 up to 1200 bar • Application-specific Agilent ChemStation

Method Scouting Wizard software for complex method scouting campaigns within a few mouse clickswithin a few mouse clicks


1200 Series HPLC-Chip/MS SystemLow-flow LC Solutions for most precious samplesp p

For reliable high sensitivity nanospray LC/MS

• Up to 3500 times more sensitive compared to conventional LC

• Fully integrated sample enrichment and y g pseparation columns, connection capillaries, fittings and nanospray emitter on polymer chip

• No peak dispersion for uncompromised chromatographic performance

“The Phosphochip is the ultimate ease of use tool for phosphopeptide analysis as it has a fully integrated microfluidic design that enables routine phosphopeptide analysis, without the hassles of clogging and plumbing in conventional nanoLC”

P f Alb t J R H k Ut ht U i it N th l dProfessor Albert J.R. Heck, Utrecht University, Netherlands


Topics






• Summary


LC’s Role in LC/MS Applications

• As inlet device for the MS

• Sample preparation step (salt removal)

• Better separation improves qualitative results(MS spectra, MSn characteristics, accurate mass)

• Better separation improves quantitative results(ion separation effects)

• Chromatographic separation offers additional parameter for specificity! Retention time!

• Allows introduction of additional detectors like ELSD, Fluorescence, RID,Radio-activity detectors, etc. to increase specificity even more.

8UHPLC to HPLC-Chip/MS

Ion Suppression

Sample matrix and coeluting compounds are affecting the ionization efficiency in LC/MS and in this way quantitative results (accuracy, precision).

4.8e5 Diphenhydraminem/z 256 174 2 5

4.8e5

1. Separation => Less ion suppression 2. Coelution => ion suppression

2.4e5

3.0e5

3.6e5

4.2e5

Diphenhydraminem/z 256.17

Ketoprophenm/z 255.10

m/z 256.17

2.4e5

3.0e5

3.6e5

4.2e5

0 20 0 30 0 40 0 50 0 60 0 70 0 80 0 90 1 00 1 10 1 20 1 30 1 400.0

6.0e4

1.2e5

1.8e5

Ketoprophenm/z 255.10

0 10 0 20 0 30 0 40 0 50 0 60 0 70 0 80 0 90 1 000.0

6.0e4

1.2e5

1.8e5

0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 1.10 1.20 1.30 1.40 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00


LC/UV vs. LC/MSWhat makes the requirements on the LC different?q

1. MS do not like high flow rates=> 2.1 mm or smaller ID column are preferred p=> Need for smaller delay volume (for speed)=> Need for smaller extra column volume (resolution)

2. MS are more sensitive=> Need for lower carryover => Need for cleaner LC=> Need for cleaner LC

Less contaminats (metals, plasticizers)=> Need for smaller injection volumes for concentrated samples


LC/UV vs. LC/MSWhat are the same requirements?q

1. Separation Efficiency from High Pressure Systems=> Speedp=> Resolution=> (Sensitivity)

2. Reproducibility and Accuracy=> Precision of Retention times (especially under ultrafast conditions) => Precision of Injection volumes=> Precision of Injection volumes


Sensitivity and Column I.D.

Column Type Inner Diameter (mm)

Sample Load (approx) Typical Flow Rate Increased

Sensitivity Applications

Analytical 4.6 0.1-1.5 mg 0.5-3 mL/min Standard separationsseparations

Solvent Saver 3.0 150-500 ug 0.3-1.5 mL/min +Save solvent, uses standard HPLC instrument

N B 2 1 50 120 0 1 0 5 L/ i

High sensitivity, limited sample,NarrowBore 2.1 50-120 ug 0.1-0.5 mL/min ++ limited sample, LC/MS, save solvent

Microbore 1.0 10-50 ug 10-100 µL/min ++++High sensitivity, limited sample, LC/MS

Capillary 0.5, 0.3 1-10 ug 1-15 µL/min +++++

Very high sensitivity, LC/MS, peptides and proteinsVery high

C/ SNano 0.1, 0.075 100-200 ng 200-500 nL/min ++++++ sensitivity, LC/MS, peptides and proteins


PROS/CONS of Low Flow LC for MS

• PROS• Support smaller I D columns• Support smaller I.D. columns• Better ionization efficiency with micro-electrospray or nano-electrospray

• CONS• Traditionally less robust, especially nanoflow LCTraditionally less robust, especially nanoflow LC• Additional expertise is required for nano-electrospray LC/MS

– Most are home-made in academic labs– Micro connections with many small fittings, capillaries, and trapping or

analytical columns


What is HPLC-Chip?

• Revolutionary microfluidicchip technology specifically

Conventional Nano LC Columnp gy p y

designed for nanosprayLC/MS

• Replaces conventional Conventional Enrichment

Electric contact

• Replaces conventional features that are now incorporated on a single chip

Conventional Enrichment Column, Capillary, Fittings,

Frits

Conventional Nanospray tip assembly and fittings

• Automated workflow using Agilent HPLC-Chip Cube MS interface and connections with Agilent Nanoflow LC

tip, assembly and fittings

Chip-Cube

with Agilent Nanoflow LCStator

Rotor


Agilent HPLC-Chip IIKey features

Colored tag

Fully integrated design• Patented Laser-ablated channels• Analytical column

RF ID Tag

g

y• Enrichment column• Micro-valve connection• Nano-electrospray tip• Micro-filters• NEW carbon ion implanted filter

Electric contact

• NEW carbon ion implanted filterPerformance enhanced• Zero dead volume for better

chromatographic performance• LC/MS sensitivity

Carbon Ion Implanted Filter

• Improved run-to-run reproducibility and chip life time

Robustness & ease-of-use• No clogging of spray needle• Plug & Play replacement• Plug-&-Play replacement• Improved surface characteristics

dramatically for optimal contact and sealingBuilt-in Micro-filter

Integrated Sprayer-tip

Integrated Enrichment Column

Integrated Nano LC Column

Inert polyimide

filterColumnColumn


Agilent HPLC-Chip/MS System

• Includes 1200 Series Capillary and Nanoflow Pumps, Micro-

• System control is through either Agilent ChemStation or p ,

well Plate Sampler with Thermostat, Chip Cube MS interface, and any Agilent 6000 Series Mass Spectrometer and

gMassHunter software

Series Mass Spectrometer and application specific HPLC-Chip


Agilent Jet Stream Technology for Regular Flow LC/MS- Increasing sampling efficiency for LC/MS

• Orthogonal spray• Efficient desolvationEfficient desolvation

– heated drying gas – nebulizing gas

ill d i– capillary design– heated sheath gas

– Improved spatial ion focusing – Improved S/N ratio

• Sensitivity increase 5- to 10-fold• Handles high LC flow (no split)


Topics



• Example Performance Data for LC/MS



• Summary


Ultra High Definition LC/MS- Optimizing all Analytical Dimensionsp g y

Signal Response

Chromatography Mass spectrometry

RobustnessRobustnessRobustness RobustnessRobustnessRobustness

Mass Spectrum


Rapid Analysis of Pharmaceutical Compounds

6 5e5

7.0e5

TOF - MS with Peak Capacity of 40 Cycle in 39 seconds

5.0e5

5.5e5

6.0e5

6.5e5

0 36s 0 42Metoprolol Verapamil

H2O/ACNFlow =1.8ml/min

5-90%B in 0.5minStop time =0.65min

80°C, ACR

3.5e5

4.0e5

4.5e5

Intensity, cps

0.36s 0.42s

Atenolol Beclomethasone-

,MS 40Hz

100-1000Da

2.0e5

2.5e5

3.0e5

0.34s

0.36s

0.36sPrimidone

dipropionate

5.0e4

1.0e5

1.5e50.36s

0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 0.55 0.60Time, min

0.0


Drug Metabolism- Coeluting Metabolites With Parent Drug

Five decades of dynamic range response in a single scan

verapamil dihydroxy metabolite of verapamil

6

p y y p

6x10

2

2.2

2.4

2.6

2.8 455.290321(M+H)+

2.68 million counts

25 counts

400 counts

1

1.2

1.4

1.6

1.8

2

desmethyl

0

0.2

0.4

0.6

0.8

1

441.274704

471.284287487.279822

(M+H)+

desmethyl metabolite

monohydroxy metabolite

0

Counts vs. Mass-to-Charge (m/z)435 440 445 450 455 460 465 470 475 480 485 490 495 500 505


6540 Ultra High Definition QTOFWide Dynamic Range PeakWide Dynamic RangeG2421A Tune Mix – m/z 3221 sec spectrum

3x10

1.6

1.8

2

2.2

2.4 325.0623021835

3x10

1.6

1.8

2

2.2

2.4 325.0623021835

PeakHeight

100

Dynamic Range: 4x10

2 5

3

3.5

4 324.0597621517

4x10

2 5

3

3.5

4 324.0597621517

0.2

0.4

0.6

0.8

1

1.2

1.4

326.2044017254

0.2

0.4

0.6

0.8

1

1.2

1.4

326.2044017254

5,000,000100

= 50,000

Peakheight

5,000,000

5x10

2

2.25

2.5

2.75 323.0585420415

5x10

2

2.25

2.5

2.75 323.0585420415

0

0.5

1

1.5

2

2.5

325.0623021835324.32499

11265326.20440

172540

0.5

1

1.5

2

2.5

325.0623021835324.32499

11265326.20440

17254

Co325 325.5 326 326.5

Co325 325.5 326 326.5

6x10

3

3.5

4322.05432

20535

6x10

3

3.5

4322.05432

20535

, ,

0.25

0.5

0.75

1

1.25

1.5

1.75

324.0597621517

325.0623021835

323.3331316656

324.3249911265

326.2044017254

0.25

0.5

0.75

1

1.25

1.5

1.75

324.0597621517

325.0623021835

323.3331316656

324.3249911265

326.2044017254

Counts vs. Mass-to-Charge (m/z)324 324.5 325 325.5 326 326.5

Counts vs. Mass-to-Charge (m/z)324 324.5 325 325.5 326 326.5

0

0.5

1

1.5

2

2.5

323.0585420415 324.05976

21517325.06230

21835326.20440

172540

0.5

1

1.5

2

2.5

323.0585420415 324.05976

21517325.06230

21835326.20440

17254

0

Counts vs. Mass-to-Charge (m/z)323 323.5 324 324.5 325 325.5 326 326.5

2183516656 11265 172540

Counts vs. Mass-to-Charge (m/z)323 323.5 324 324.5 325 325.5 326 326.5

2183516656 11265 17254

0

Counts vs. Mass-to-Charge (m/z)322 322.5 323 323.5 324 324.5 325 325.5 326

0

Counts vs. Mass-to-Charge (m/z)322 322.5 323 323.5 324 324.5 325 325.5 326

+ESI Scan (#27) Frag=180.0V 2GHz_accF.d +ESI Scan (#27) Frag=180.0V 2GHz_accF.d


1290 LC for fast/sharp peaks that require fast MRMs6430/6460 run 200 MRMs/sec, needed for 1 sec peaks, p


Eight minute analysis of 224-pesticides 500 ppt using the new Agilent 1290 Infinity LC & 6460 pp g g y

Retention Time Window = 12 sec

6460 Triple Quad LC/MS Systemwith dynamic MRM 12 sec

Peak widths ~ 1 secCycle time: 100 msecMin dwell: 2.5 msec

with dynamic MRM


Spiked Plasma StudiesUHPLC Method with 1290 Infinity LC InjectorUHPLC Method with 1290 Infinity LC Injector

• Zorbax Eclipse Plus C18 RRHD (50 x 2.1 mm; 1.8 µm)• T l = 50°CTcolumn 50 C• A: 5 mM NH4OOCCH3

• B: ACN• flow = 0 5 mL/min

Compound NamePrecursor Ion

(FV)Prod Ion 1

(CE)

Fluticasone Prop 501.2 (119) 293.2 (12)• flow = 0.5 mL/min

T(min)%B0 501.3 701.4 901.7 901.8 50R ti 2 + 1 i t• Run time = 2 + 1 min post

• MS: 6460 Triple Quad


Linearity and LLOQFl ti P i t- Fluticasone Proprionate

nses

Res

po

Concentration range: 0.05 – 50 ng/mLLLOQ: 50 pg/mL = 50 fg abs on column (S/N > 5 / FDA

guidelines for precision and accuracy)g p y)Noise = 5 x RMS: 0.65-0.85 & 1.1-1.3)


LLOQ and Carryover- Plasma: data\Fluticasone\100401a\CTC\PlasmaPlasma: data\Fluticasone\100401a\CTC\Plasma

+ MRM (501.2 -> 293.2) S5-r003.d1x10

5.8

6

6.2

*Fluticason Proprionate

+ MRM (501.2 -> 293.2) blank Post S11_2.d1x10

6

6.2

+ MRM (501.2 -> 293.2) S11_3.d4x10

1.1

1.2

1.3

1.4Fluticason Proprionate

LLOQ Highest std Zero carry

4.8

5

5.2

5.4

5.6

5

5.2

5.4

5.6

5.8

0 4

0.5

0.6

0.7

0.8

0.9

1LLOQ50 pg/mL

Highest std injected

50 ng/mL

Zero carry over

0 5 0 6 0 7 0 8 0 9 1 1 1 1 2 1 3 1 4 1 5

3.8

4

4.2

4.4

4.6

4

4.2

4.4

4.6

4.8

Acquisition Time (min)0.5 0.6 0.7 0.8 0.9 1 1.1 1.2 1.3 1.4 1.5

-0.1

0

0.1

0.2

0.3

0.4



3.8Acquisition Time (min)


Topics






• Summary


What is...?- PK

Typical plasma level curve and parameters of interest


What is...?- Pre-clinical PK trials

Discovery Preclinical Development

Clinical Trials

ManufactureQA/QC

Pre-clinical PK

Cost // Time

Pre-clinical PK• Animal PK studies (typically mice / rats)• Quantitative LC/MS to monitor drug absorption and

decay in blood plasmadecay in blood plasma• PK data for estimation of safe starting dose for clinical

trials in humans

Challenge• Nowadays, early in drug discovery phase to fail non-

drug like compounds early, fast and therefore cheap g p y, p• Requires fast sample turnaround due to the large

number of new potential drug candidates


What is...?- Cassette dosing (a.k.a. N-in-1 & cocktail dosing)Cassette dosing (a.k.a. N in 1 & cocktail dosing)

Cassette dosingCassette dosing• Widely used to accelerate in-vivo PK screenings• Cocktail of drugs administered to animal• PK testing in parallel means

- fewer rodents- less animal handlingg- less sample prep - reduced analysis

• Commensurate to ethics and cost beneficial• Commensurate to ethics and cost beneficial


What is...?- Dried Blood Spot (DBS) technology- Dried Blood Spot (DBS) technology

Sample prep w/ filter cards (e.g. FTA Micro Elute; Whatman)

• Spot full bloodp• Dry room temperature• Punch discs (manual // automated)• Transfer extraction tubes // well-plateTransfer extraction tubes // well plate• Add solvent organic containing ISTD• Extraction ultra sonication / skake / vortex• Spin• Spin• Inject

Dr. Stephan BuckenmaierResearch Scientist

Agilent Technologies USA


Agilent Technologies, USA

Why using DBS...?- Great interest in DMPK area due to range of advantages

• Small sample volume (10-20 µL) facilitates pediatric studies Less invasive finger prick rather than venous cannula

G eat te est a ea due to a ge o ad a tages

• Less invasive finger prick rather than venous cannula– improves recruitment for clinical studies

• Simple storage and transport e g to remote contract laboratoriesSimple storage and transport e.g. to remote contract laboratories– no freezers / dry ice / special biohazard arrangements (antimicrobial)

• Cost – reduced rodent size & number in pre-clinical studies– reduced amount of active drug / PK-study

• Data quality – serial bleeding vs. composite

Ethical benefit• Ethical benefit


REACH (Registration, Evaluation, Authorisation and Restriction of Chemicals)and Restriction of Chemicals) Euro Biotech News, No. 9-10, Vol. 8, September 2009

• Pre registered around 140 000 chemical substances for safety testing• Pre-registered around 140,000 chemical substances for safety testing within REACH (initial estimate was 29,000)

• Require around 54 million test animals in the next decade• Cost of REACH to EUR9.5bn – six times the original estimate• 3R (replacement, refinement and reduction) experts indicated that the

majority of the animal tests would also have only limited valuej y y

Solutions??? DBS?


Goals // Challenges

Utilize Dried Blood Spot (DBS) procedure to extract a cassette of drugs from miniscule volumes of blooddrugs from miniscule volumes of blood

• Combining both approaches to maximize ethical and cost benefits• chromatographic separation quality in high throughput mode

d t ti iti it• detection sensitivity

Base of operation• Typical run time in PK: 3 – 5 min• ≥ 6 min for cassette dosing (typically 5 compounds)• Typical LLOQ for parent 1 ng/mL• Low level metabolites (10% of parent) as suggested by FDA


Experimental- Cassette spiked into rat blood

Verapamil (V)

- Cassette spiked into rat blood

OCH3

Dextromethorphane (DM)

≠

Dextromethorphane (DM) Methoxy Verapamil (MV)

≠≠

Protriptyline (P) Imipramine (T) Trimipramine (T)Amitriptyline (A)


1200 Series RRLC- Goal: Run time ≤ 6 min

Conditions• Zx Eclipse Plus C18 RRLC (50 x 2.1 mm; 1.8 µm)

- Goal: Run time ≤ 6 min

• Tcolumn = 50°C• ACN or MeOH [0.1% FA] gradient• flow = 0.5 mL/min

Advantages MeOH• Price (purchase & disposal)• Less hazardousess a a dous• Environmental friendliness

Resultd R h• α - and Rs- change

• Higher back pressure w/ MeOH [pmax ~ 420 bar]

• Run time conditions achieved *10 column volumes ~ 3.4min at flow 0.5mL/min


1290 Infinity LC - Increase flow to yield extremes in speedy p

High speed chromatography plus separation quality• Short RRHD column (50 x 2.1 mm ID, 1.8 µm)• Minimal gradient delay (e.g. pump 10 µL)• Flow (2 mL/min at 1,200 bar)


1290 Infinity LC - Increase flow to yield extremes in speedy p

High speed chromatography plus separation quality• Short RRHD column (50 x 2.1 mm ID, 1.8 µm)• Minimal gradient delay (e.g. pump 10 µL)• Flow (2 mL/min at 1,200 bar)

2 mL/min

Van Deemter plot w/ particle size

2 mL/min

2 mL/min


Experimental- Goal: Go fast, maintain separation qualityGoal: Go fast, maintain separation quality

Conditions• Zorbax Eclipse Plus C18 RRHD (50 x 2.1 mm; 1.8 µm)• Flow = 0.60 - 1.75 mL/min• B: MeOH [0.1%FA]

T(min) %B0 400 401 601.2 901.7 901 9 401.9 40

R ltResults• p-increase [pmax ~ 1160 bar @ 1.75 mL/min]


Chromatographic Results- Cassette analysis of DBS extracts 0.05 – 500 ng/mL bloodCassette analysis of DBS extracts 0.05 500 ng/mL blood

Chromatographic performanceSeparation of seven drugs• Separation of seven drugs

• RTmax (ISTD) ~ 42 seconds• Peak w0.5 ~ 1 second


Linearity Results- Cassette analysis of DBS extracts 0.05 – 500 ng/mL bloodCassette analysis of DBS extracts 0.05 500 ng/mL blood

LinearityC lib ti ( 3 i j ti / d t i t)• Calibration curves (n=3 injections / data point)Relative Response = Area (X) / Area (ISTD)

• Correlation factors (R2) approach unity for all compounds in the cassettecompounds in the cassette


LLOQ Results- Cassette analysis of DBS extracts 0.05 – 500 ng/mL bloodCassette analysis of DBS extracts 0.05 500 ng/mL blood

LLOQ• Criteria based ion FDA guidelines• Criteria based ion FDA guidelines

Response > five times resp (blank)Precision (n ≥ 5 inj.) < 20 %RSDAccuracy range 80 – 120 % y g


HPLC-Chip/MS for DMPK??

The ideaAchieve substantial - savings

x

x

xx

xPK-profile

timexx

Mice cheaper to purchase, keep and breedMice require less active pharma ingredient than dogs or rats...

The challengeMice can only donate so much bloodSerial bleeding increases PK-data quality very limited samplesSPE to increase detection sensitvity not an efficient option

The Chip solutionMi i t i ti b d iti it ll t h dl ll t l titi

UHPLC to HPLC-Chip/MS

Miniaturization based sensitivity allows to handle smallest sample quantities

45

HPLC-Chip/MS for Small Molecules

Trapping-column: 500nl

Ultra-high Capacity HPLC-Chip (UHC-Chip)

pp gNano-column: 150mm x 75µm IDPacking: Zorbax SB C18, 5µm (80Å)

Separation column 150mm x 75µm ID

Pre-concentrate a wide range of polarities

UHC-Chip should… 500nL enrichment column

Pre concentrate a wide range of polaritiesPossess high loading capacityDemonstrate retention time and peak area precision/repeatability/reproducibilityEven enhance sensitivity-performance of the 6410 QQQ mass spectrometer

UHPLC to HPLC-Chip/MS

Even enhance sensitivity performance of the 6410 QQQ mass spectrometer

46

Potentially simplified Sample-PreparationBl d FTA® t ti & l t i i it tiBlood FTA®- extraction & plasma protein precipitation

Spot 10µl blood spiked with nortriptyline onto FTA® Elute Micro Cards (Whatmann)

Disks + 20µL ACN/H2O 75:25

Dilute w/ H2O (df =7.5)

15min @ 15,000rpm

Mix (30min)

Few min @ 4,000rpm

Protein Precipitation Centrifugation & transfer of supernatant

Reconstitution300µL ACN Loading mobile phase

SpeedVac Inline filterUpchurch 1µm Titanium

100µL plasma + imipramine 10min @ 13,000rpm


UHC-Chip for high-sensitivity Chip/QQQ in DMPK

EIC QuantifiersImipramine

logP 4 2

Atropine102

Sensitivity

UHC Chi /QQQ d li 100070802\1pg_13

Atenolol

logP 0

logP 4.2

Metoprolol

•UHC-Chip/QQQ delivers 100xincrease in sensitivity comparing to the conventional electrospray at higher flow rate

104

g

Selectivity

•UHC-Chip (ultra-high capacity HPLC-Chip) solution can be used to

Overlay of chrom on top and EIC from

070821\1pg_16Imipramine

AtropineHPLC Chip) solution can be used to analyze compounds with a wide range of hydrophilicity

Atenolol Metoprolol


Q f

Early R&D-Prototype Results

Quantitative enrichment of samples in 70% ACN

Elimination of dilution step “and ” increase of inj.-V

1 … Atenolol2 … Nadolol3 … Atropine4 … Metroprolol

2

7

)

TrimipramineR2 = 0.9924

5 … Labetalol6 … Propranolol7 … Imipramine8 … Trimipramine

1

46

8

P =

4.2

tens

ity (x

107

eak

Are

a

PropranololR2 = 0.9911

Atenolol1

3

5

logP

= 0 lo

g PIn

4µl inj.-VPe Atenolol

R2 = 0.9961

AtropineR2 = 0.9966

Acquisition Time (min) 2 4 6 8

Injection Volume (µl)

R 0.9966

This would imply: 0.05ng/mL / [1.3 x 7] ~ 5pg/ml out of 10µl of blood


Topics






• Summary


Intact mAb Analysis (HPLC-Chip/MS): Intact and Deglycosylated

G0F/G0F

Deglycosylated

Intact mAb

Δ2890.81

Δ1444.87

4x10

5

6

7

8145924.41

DeglycosylatedNo glycan-attached species

1

2

3

4

146272.08

g y y(sugar groups removed enzymatically)

0

Counts vs. Deconvoluted Mass (amu)144500 145000 145500 146000 146500 147000 147500 148000 148500 149000 149500


Summary – Intact mAb Characterization Results from HPLC-Chip Accurate Mass QTOF Analysesfrom HPLC Chip Accurate Mass QTOF Analyses

Mcalc Mexp Error (ppm)

Intact major glycoform 148,811.95 148,812.81 5.8

deglycosylated 145,923.24 145,924.41 8

Light chain 23,746.63 23,746.50 5.5

H h iHeavy chainmajor glycoform 50,675.47 50,675.58 2.2

Fcmajor glycoform 52,755.62 52,755.64 0.4j g y

FAB 48,046.18 48,046.09 1.8


Agilent 1290 + 6530 UHD Q-TOF for Rapid Peptide MappingMapping

• Chromatographic peak• Chromatographic peak width (half-height) 0.3-0.8 seconds

• MS acquisition (300-3000) at 10 spectra/sec in high resolution modein high resolution mode

• 98.8% sequence coverage in 1.5 min!coverage in 1.5 min!


1.5 Minute LC/MS/MS Analysis of Serotransferrin DigestDigest

81% sequence coverage and 93 unique peptides in 1 5 min!!unique peptides in 1.5 min!!

• Red indicates matched peptides• For transferrin, the first 19 amino acids are the signal peptide


On-Chip Glycan Characterization in 10 MinutesNew N-GlycoPRO Chip

mAb sample

y p

Enzymatic N-glycan Release 6 seconds

mAb sample

Ex

LC/Fluorescence

10 min

GlycoPRO-Chip-MS

MALDIMS

N-glycan Recovery

N-Glycan Concentration

6 seconds

6 seconds

perimenta Concentration

HPLC Analysis 2 minutes

al Time

1 day

//

//

TOF MS Detector 2-3 days

//

Time:10 minutes


On-chip Deglycosylation and Analysis of Intact Antibodies 4x10 Intact Antibody Prior to DeglycosylationAntibodies 4x10

2

3

4

5

6

149089.58

149253.47

149415.31

Intact Antibody Prior to Deglycosylation

0

1

2148570.91

4x10

7

83 sec. deglycosylation

• Intact mass of heavily modified proteins is difficult to measure

2

3

4

5

6

7

1 glycan removed146202.21

147645.93

147806.94

147962.53

difficult to measure.

• By deglycosylating the protein first the

0

1

4x10

5

6

Deglycosylated mAb

6 sec. deglycosylation

protein first, the accurate protein mass is measureable.

0

1

2

3

4 Both glycans removed

0

Counts vs. Deconvoluted Mass (amu)145500 146000 146500 147000 147500 148000 148500 149000 149500


N-GlycoPRO Chip Enables Fast Separation of Isomers Using an Extended GradientIsomers Using an Extended Gradient

G2 t tG2 structure

Potential immunogenic

1786.650 amug

G2 structure


Comparison of Chip Data to CE and MALDI Methods

70

50

60

70

[mol%]

MALDI‐MS (N=34)

CE‐LIF (N=43)

Agilent Glycan Workflow Chip (N=50)

30

40

50

ar distribut

ion

g y p ( )



10

20

Relativ

e mola

0

Man‐5 G0‐GlcNAc G0‐F G0 G1 G2


Summary

• Importance of separation in LC/MS

Recent advances in separation for LC/MS• Recent advances in separation for LC/MS• UHPLC for faster and better resolution• HPLC-Chip/MS for ease-of-use nano-electrospray MS• HPLC-Chip/MS for ease-of-use nano-electrospray MS

• Dried Blood Spot is compatible with both UHPLC and HPLC-Chip/MS

• UHPLC and HPLC-Chip/MS methods are both suitable for biopharmaceutical analysis


Thank you!!!


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