analysis, purification and validation of target compounds

Analysis, purification and validation of target

compounds

Covering the complete compound generation workflow with Agilent 1200 Series LC/MS solutions

Udo Huber Product Specialist

October 31, 2007

Overview

• The compound generation workflow

• Analytical and preparative tasks in the compound generation workflow

• Agilent solutions for:

• Walk-up systems

• Fast result generation

• High sample throughput

• Sample purification

• Compound confirmation

From building blocks to hits – the compound generation workflow

Buildingblocks HitTarget

compounds

Compoundlibrary

Synthesis Screening

Storage

Analytical and preparative tasks


compounds

Compoundlibrary

Synthesis

Storage

QC PurificationControl ofreaction outcome

Final QCCharacterization

Identity & purity

Prep LC/MS

Routine randominspection

Purification

Screening

Identity & purity

Standard can be any compound with nitrogen (adjacent

nitrogens)

Without standard compound 3D calibration is

required (inverse gradient)

Requires standard of compoundAbsolute

Only compounds with nitrogen are

detected

Non-linear response, response

depends on composition of mobile phase

Area % report, response is compound

dependent, can be zero

Relative

CLNDELSDUV (DAD)Purity

Standard can be any compound with nitrogen (adjacent

nitrogens)

Without standard compound 3D calibration is

required (inverse gradient)

Requires standard of compoundAbsolute

Only compounds with nitrogen are

detected

Non-linear response, response

depends on composition of mobile phase

Area % report, response is compound

dependent, can be zero

Relative

CLNDELSDUV (DAD)Purity

Identity: SQ mass + knowledge of compound structureAccurate mass (TOF), NMR, IR, etc. for final QC of hits

Compound generation – facts

Medium or large numbers of compoundscertain level of automation is required

Instrument users are often synthetic chemistsnot chromatographers, system must be simple to use

Structure and mass of building blocks/compounds known

Non-regulated environment

Analysis of compounds – the basic solution

The workhorse: 1200 Series LC system with 6100 SQAgilent ChemStation

• Binary or quaternary pump with degasser

• Standard or well-plate autosampler

• Column compartment• DAD (MWD)• 6110, 6120 or 6130 SQ

The Agilent 6100 Series single quadruple

Research GradeHigh Throughput6140

m/z 10 – 1350 mass range1 pg sensitivity spec (50 pg scan)Native 2500 u/s scan speed10000 u/sec scan speed1.4 MHz quadrupolePOS/NEG and divert valveMatched to 1200 RRLC performance

Entry Level6110

m/z 10-1500 mass range10 pg sensitivity2500 u/s scan speed1 MHz quadrupoleOptional Automated Divert Valve

Mid Range6120

m/z 10-1500 mass range10 pg sensitivity2500 u/s scan speed1 MHz quadrupoleAutomated Divert Valve (MMS)POS/NEG switching

Research Grade6130

m/z 2 – 3000 mass range1 pg sensitivity spec (50 pg scan)Native 2500 u/s scan speedFast scanning mode 5250 u/sec1.0 MHz quadrupolePOS/NEG and divert valve

Upgrade kitG2735F

Upgrade kitG2735E

Upgrade kitG2735G

Analysis of compounds – Easy Access

• Easy Access Software allows chemists untrained in detailed operation of the instrument data system and possibly unskilled as chromatographers to run their own samples safely.

• Same software interface for LC, LC/MSD and LC TOF.

• It is not a software for “inexperienced users”! It is a software for a system-user/system-administrator environment!

“The system users are chemists who need results, not chromatographers who want to play with the instrument or tweak the software!”

External tray for standard autosampler SL

Vials discarded here on completion (with optional delay)

Chemists put up to 17 vials in external tray. Sampler arm moves them inside.

E-mail notification of sample results

Attached Data Browser file

Data Browser

Easy Access in the compound generation workflow


compounds

Compoundlibrary

Synthesis Screening

Storage




Purification

Analysis of compounds – fast chromatography

Medium or large numbers of compoundsMore compounds require faster analysis:The 1200 Series RRLC system with 6140 SQ MS

• Binary pump SL with degasser

• High-performance autosampler SL

• Column compartment SL• Sub-2-µm particle column• DAD SL (MWD SL)• 6140 SQ


The 1200 Series RRLC system with 6210/6220 TOF MSAdditional accurate mass information gives higher confidence of target compound



• Column compartment SL• Sub-2-µm particle column• DAD SL (MWD SL)• 6210/6220 TOF


In a walk-up environment the number of samples is usually low.With fast chromatography chemists do not have to wait a long time to get their results and can continue work earlier:The 1200 Series RRLC system with 6140 SQ MS


• Standard autosampler SL with external tray

• Column compartment SL• Sub-2-µm particle column• DAD SL (MWD SL)• 6140 SQ• Easy Access software

Analysis of compounds – matching capacity to throughput

Medium or large numbers of compoundsMore compounds require higher sample capacity:The 1200 Series (RRLC) system with CTC PAL


• HTS or HTC PALwith 600 bar injection valve

• Column compartment SL• Sub-2-µm particle column• DAD SL(MWD SL)• 6140 SQNow compatible with Easy-Access

Analysis of compounds – matching capacity to throughputMedium or large numbers of compoundsMore compounds require higher sample capacity:The 1200 Series (RRLC) system with sample capacity extension


• High-performance autosampler SLwith sample capacity extension

• Column compartment SL• Sub-2-µm particle column• DAD SL (MWD SL)• 6140 SQ

High throughput and RRLC in the compound generation workflow


compounds

Compoundlibrary

Synthesis Screening

Storage




Purification

The Agilent 1200 Series purification system

• 2 preparative pumps• Dual-loop autosampler• ZORBAX preparative

column• MWD (DAD)• 1-3 fraction collector PS• 6110, 6120 SQ

with active splitter

Preparative HPLC is the method of choice for compound purification in compound generationThe 1200 Series purification system with 6100 SQ MS

Mass-based fraction collection

Totals formulas of target compound known

Mass of target compound known

Mass-based fraction collection

Advantages:• Small number of fractions per

sample, often only one fraction per sample

• Only target compound collected (or compounds with the same mass).

• No need to analyze fractions to determine which one contains the compound of interest.

Disadvantages:• Target mass must be known• Purity of fractions slightly lower

than for UV based collection.• System is more expensive• Seems to be more complicated

(splitter, etc.)

Sample purification


compounds

Compoundlibrary

Synthesis Screening

Storage




Purification

Final characterization of compounds

High-resolution LC gives confidence to separate most compounds.High mass accuracy of TOF MS gives high confidence of compound identity.The 1200 Series RRLC system with 6210/6220 TOF MS



• Column compartment SL• Sub-2-µm particle column• DAD SL (MWD SL)• 6210/6220 TOF

How much accuracy is needed?

Example:Reserpine (C33H40N2O9) has a protonated ion at 609.28066Single quad reports mass to +/- 0.1 = 165 ppm

Number of possible formulas using only C, H, O & N:165 ppm 20910 ppm 135 ppm 73 ppm 42 ppm 2

+ information about totals formula

Typical performance of TOF instrumentMass error histogram of the analysis of 140 real screening compounds under ultra-fast LC conditions (90 s cycle time 1000 samples/day):

0

5

10

15

20

25

30-5

.0

-4.5

-4.0

-3.5

-3.0

-2.5

-2.0

-1.5

-1.0

-0.5 0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

91 % within ± 2.0 ppm

ppm

n pe

r 0.2

5 pp

m

71 % within ± 1.0 ppm

Summary

• The compound generation workflow

• Analytical and preparative tasks in the compound generation workflow

• Agilent solutions:

• Easy Access software for walk-up systems

• RRLC for fast results or higher sample throughput

• Solutions for high sample capacity – HTC and HTS autosampler and sample capacity extension

• Preparative HPLC – Agilent purification system

• Reliable compound confirmation – Agilent TOF MS

analysis, purification and validation of target compounds

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