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MEDICINAL CHEMISTRY APPLICATIONS BOOK

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Page 1: Waters Medicinal Chemistry Applications Book · 3 The Role of liquid ChRomaTogRaphy and mass speCTRomeTRy in mediCinal ChemisTRy “ Medicinal chemistry is a scientific discipline

MEDICINAL CHEMIST RY AP PLICATIONS BOOK

Page 2: Waters Medicinal Chemistry Applications Book · 3 The Role of liquid ChRomaTogRaphy and mass speCTRomeTRy in mediCinal ChemisTRy “ Medicinal chemistry is a scientific discipline

Introduction .................................................................................................................................................................................................3

The Role of LC and MS in Medicinal Chemistry .........................................................................................................................................5

System Management for a High Throughput Open Access UPLC/MS System Used During the Analysis of Thousands of Samples ............................................................................................................. 11

OpenLynx Open Access ...........................................................................................................................................................15

New Tools for Improving Data Quality and Analysis Time for Chemical Library Integrity Assessment .............................23

Scaling a Separation from UPLC to Purification Using Focused Gradients ...........................................................................29

Purification Workflow Management ........................................................................................................................................33

Making a Purification System More Rugged And Reliable ....................................................................................................39

Application of MS/MS Directed Purification to Identification of Drug Metabolites in Biological Fluids ............................45

Evaluating the Tools for Improving Purification Throughput .................................................................................................51

A Novel Approach for Reducing Fraction Drydown Time ......................................................................................................57

ProfileLynx Application Manager for MassLynx Software: Increasing the Throughput of Physicochemical Profiling ................................................................................................................................63

An Automated LC/MS/MS Protocol to Enhance Throughput of Physicochemical Property Profiling in Drug Discovery ......................................................................................................................................65

Synthetic Reaction Monitoring Using UPLC/MS .....................................................................................................................71

ACQUITY UPLC System: Time and Cost Savings in an Open Access Environment .............................................................73

SCREENING

CONFIRMATION

PURIFICATION

PROFILING

OPTIMIZATION

Page 3: Waters Medicinal Chemistry Applications Book · 3 The Role of liquid ChRomaTogRaphy and mass speCTRomeTRy in mediCinal ChemisTRy “ Medicinal chemistry is a scientific discipline

3

T he Role of l iquid ChRomaTogRaphy and mass speCT RomeT Ry in mediC inal ChemisT Ry

“ Medicinal chemistry is a scientific discipline at the intersection of chemistry and pharmacy involved with designing,

synthesizing, and developing pharmaceutical drugs. Medicinal chemistry involves the identification, synthesis and

development of new chemical entities suitable for therapeutic use.”

– Wikipedia.coM

The objective of medicinal chemistry is to design and discover com-

pounds that offer the potential to become beneficial – and profitable

– therapeutic drugs. confidently confirming the identity and quality

of these new chemical entities is a major challenge, particularly

when labs are asked to maximize throughput and efficiency – and to

manage all the data generated by a variety of systems and users.

Medicinal chemistry is also an iterative process that demands

rapid turnaround times. High throughput liquid chromatography/

mass spectrometry (Lc/MS), together with advanced data-handling

software, has become the standard technique for drug discovery

compound identification and purification, addressing needs for high

throughput screening, optimization, and physicochemical property

profiling.

Waters Ultraperformance Lc® (UpLc®) technology is providing a sea

change in capacity for medicinal chemistry labs. UpLc uses sub-

two-micron column particle sizes to produce faster, more sensitive

and high-resolution separations. our UpLc systems are available

with fast-scanning detectors, both optical and mass, and can be

easily controlled by software that facilitates sample analysis in

open-access laboratory environments.

in this applications book, we look at a variety of system solutions

that address the unique challenges of medicinal chemists in five

key areas.

n in screening, we will demonstrate the use of high UpLc

throughput and fast-scanning MS to obtain high quality

and comprehensive data about compounds in the shortest

possible time.

n For Compound Confirmation, we will show how an open access

interface, used with UpLc technology and advanced detection,

enables chemists with minimal instrument training to determine

the identities of known compounds, to rapidly identify un-

knowns, and to characterize complex sample components.

n in purification, we provide several examples on how chemists

can use UpLc along with efficient time-saving techniques to

dramatically increase throughput.

n in Compound profiling, we illustrate an automated UpLc/MS/

MS protocol that not only allows for automated MS method de-

velopment and data acquisition, but also allows data generated

from multiple assays to be automatically processed by a single

processing method.

n in optimization, we will show how chemists were able to quickly

and easily monitor their reactions, noting the relative amounts

of starting materials and products by using a walkup UpLc/MS

system.

Page 4: Waters Medicinal Chemistry Applications Book · 3 The Role of liquid ChRomaTogRaphy and mass speCTRomeTRy in mediCinal ChemisTRy “ Medicinal chemistry is a scientific discipline

inT RoduCT ion

confirming the identity and quality of new chemical entities is a

major challenge facing the pharmaceutical industry. Maximum

efficiency is essential for laboratories challenged by throughput

requirements and the management of data from a variety of

systems and users.

Liquid chromatography with mass spectrometry has become the

standard technique for confirming the identity and purity of drug

discovery compounds to support high throughput screening (HTS),

optimization, and physicochemical property profiling of these com-

pounds. Medicinal chemistry is an iterative process and requires rapid

turnaround times. High throughput solutions together with advanced

data handling software must be employed.

in this application note, we look at various solutions, including

sub-2 µm column particle sizes, fast scanning mass spectrometers,

and new software to assist the medicinal chemist in five key areas:

n Screening

n confirmation

n purification

n compound profiling

n optimization

meT hods and disCussion

screening

it is important to verify the identity and purity of a compound before

early activity studies. chemists need to be sure they have synthe-

sized the expected compound. Large numbers of compounds may be

created, so it is necessary for this screening to be high throughput.

Because only a small amount of material is synthesized, the screening

must also consume as little material as possible, while generating a

diverse amount of information.

T H E RO L E O F L IQU I D C H ROMAT OG R A P H Y A N D MA S S S P EC T ROM E T RY IN M E D IC INA L C H EM IS T RY

darcy Shave, paul Lefebvre, and Marian Twohig Waters corporation, Milford, Ma, U.S.

Samples were analyzed on a Waters® acQUiTY UpLc® System

with a Sample organizer. The column was an acQUiTY UpLc BeH

c18 (1.7 µm, 2.1 x 50 mm) run at 30 °c. The injection volume was

5 µL. compounds were separated using a generic water/acetonitrile

gradient that was 1.1 min long.

detection was done with an acQUiTY UpLc photodiode array

(pda), acQUiTY UpLc evaporative Light Scattering (eLS), and SQ

Mass detector with an eSci® source for eSi/apci switching. plates

were logged into and processed with the openLynx™ open access

application Manager for MassLynx™ Software.

By using an acQUiTY UpLc System with the Sample organizer, we

were able to analyze 3840 samples in under 7 working days on a

single column. on a traditional HpLc system, this would take approxi-

mately 27 working days, assuming a 10-minute run time.

The eSci source on the mass spectrometer allowed the chemist to

gather data in both electrospray and apci (with positive/negative

switching) modes during the same injection. in this way, the maximum

amount of data was generated with a minimal amount of sample.

Figure 1. The ACQUITY SQD with the Sample Organizer plus PDA and ELS detectors.

Page 5: Waters Medicinal Chemistry Applications Book · 3 The Role of liquid ChRomaTogRaphy and mass speCTRomeTRy in mediCinal ChemisTRy “ Medicinal chemistry is a scientific discipline

6

The open access interface allowed the user to log in the sample

plates while providing a minimal amount of information. a series

of methods, each including gradient conditions, MS conditions, and

processing parameters, was designed by the system administrator.

The user simply chose a method from this list, imported their sample

lists, and placed their microtitre plates in the indicated positions.

The samples were then analyzed and the data was processed. once

processing was finished, the data was automatically copied to a

file storage pc. From here the users could do further processing, if

desired. a report file was also generated from the processed file and

converted to pdf. This facilitated storage of the results in a database.

Confirmation

exact mass experiments permit elemental composition determi-

nations of unknowns or confirmation of a suspected elemental

composition. This allows the medicinal chemist to confirm identities

of known compounds, to rapidly identify unknowns, and to character-

ize complex sample components.

Samples were analyzed on an acQUiTY UpLc System. The column

was an acQUiTY UpLc BeH c18 (1.7 µm, 2.1 x 50 mm) run at 30 °c.

The injection volume was 5 µL. compounds were separated using a

generic water/acetonitrile gradient that was 1.1 min long.

detection was done with an acQUiTY UpLc pda and an LcT premier™

Xe Mass Spectrometer with an eSci source for eSi/apci switching.

Samples were logged into the system using openLynx open access

and processed with MassLynx openLynx with i-FiT™ exact mass

processing.

Figure 2. OpenLynx OALogin plate login wizard.

a fast generic liquid chromatographic method was designed to provide

excellent selectivity without compromising either chromatographic

resolution or speed of analysis. To obtain such an analytical method,

UpLc® in conjunction with oa-ToF MS detection was employed. With

this analytical system, identification of the anticipated samples,

isomers, and possible impurities with mass accuracy deviations less

than 5 ppm from the actual were obtained using LockSpray™. With

such high accuracy data, the calculation of elemental compositions

for each of the analytes was possible.

Subsequent elemental composition results were produced using the

i-FiT algorithm, which takes into account the distribution of the spec-

tral isotopes for the compounds of interest and employs novel data

interpretation to simplify results lists returned.

The open access interface allowed the medicinal chemist to log in

the samples while providing a minimal amount of information. The

results, including a pdf report showing the most probably elemental

compositions, were then made available to the chemist.

Page 6: Waters Medicinal Chemistry Applications Book · 3 The Role of liquid ChRomaTogRaphy and mass speCTRomeTRy in mediCinal ChemisTRy “ Medicinal chemistry is a scientific discipline

7

purification

Having a pure building block is important for controlling the syn-

thetic reactions and successfully making a pure target. a pure target

is critical for understanding the results of screening and building

quality structure/activity relationship (SaR) information.

Reverse-phase HpLc has been successfully applied to the different

aspects of the medicinal chemist’s process. it is capable of purifying

milligrams to multiple grams in a single system, and can be con-

figured to automatically process hundreds of samples. The results

can provide high purity and recovery of the desired compounds with

minimal user intervention.

Samples were analyzed on a Waters autopurification™ System,

including a 2545 Binary Gradient Module, 2767 injector, and

collector, and a System Fluidics organizer (SFo). The compounds

were purified on an XBridge™ prep c18 odB™ column (5 µm,

19 x 50 mm) run at room temperature.

detection was done with 2996 pda, eLS, and 3100 mass detectors.

Fraction collection and processing was done with the FractionLynx™

application Manager. compounds were separated using 5-minute

gradients that were chosen by the autopurify™ functionality of

FractionLynx.

Figure 3. MS and UV chromatograms showing targeted mass and impurities.

a rapid Lc/MS method was developed for the analysis of a medicinal

chemistry library. The MS data confirmed the presence of the target

compound and its retention time from a high resolution Lc separation

with a 1-minute cycle time. The retention time corresponded to a

percent organic solvent at which the compound eluted.

Based on this correspondence, a focused purification method for a

19 mm i.d. column with 5 micron particles was selected to maintain

the analytical resolution. The isolated target was then separated by

Lc. The original analytical methodology was then used to determine

the new purity for each compound collected.

By logging in their samples just once, the medicinal chemists were

able to get a purified product along with reports showing the initial

and final purities.

Compound profiling

in an effort to avoid clinical failures, there is an emphasis across the

pharmaceutical industry on examining pharmacokinetic and safety

profiles earlier in the drug discovery process. assays are developed

in order to select compounds with the highest probability of becom-

ing successful drugs based on preferred pharmacological properties.

This step includes extensive testing for the absorption, distribution,

metabolism, excretion, and toxicity (adMeT) and physicochemical

properties of a compound.

Samples were analyzed on an acQUiTY UpLc System with a Sample

organizer. The column was an acQUiTY UpLc BeH c18 (1.7 µm, 2.1

x 50 mm) run at 30 °c. The injection volume was 5 µL. compounds

were separated using a generic water/acetonitrile gradient that was

1.1 min long.

detection was done with an acQUiTY UpLc pda, a acQUiTY UpLc

eLS and a Quattro premier™ Xe Mass Spectrometer with an eSci

source for eSi/apci switching. MS conditions were optimized using the

Quanoptimize™ application Manager. The samples were processed

using the profileLynx™ application Manager. properties analyzed

included solubility, logp, microsomal stability, and cHi.

Page 7: Waters Medicinal Chemistry Applications Book · 3 The Role of liquid ChRomaTogRaphy and mass speCTRomeTRy in mediCinal ChemisTRy “ Medicinal chemistry is a scientific discipline

8

Figure 4. ProfileLynx browser showing results of solubility experiment.

early screening of physicochemical properties (pp) is an integral

process for modern drug discovery. Typical pp profiling practices

include properties such as solubility, stability (pH and metabolic),

permeability, integrity, etc. The critical factor to consider in pp profil-

ing is throughput. The bottlenecks to throughput include MS method

optimization for a large variety of compounds and data management

for the large volume of data generated.

an automated UpLc/MS/MS protocol was developed that not only

allowed for automated MS method development and data acquisition,

but also allowed data generated from multiple tests to be processed

by a single processing method, all in an automated fashion. as a

result, the physicochemical profiling process was significantly simpli-

fied and throughput increased.

The column manager bypass channel allowed users to easily switch

to direct flow injection analysis for compound optimization without

sacrificing one of the column positions. chemists can choose the

optimal conditions and chemistry for their compounds as the column

manager is a thermostat-controlled oven with temperature regulation

from 10 to 90 °c and has automated switching for four columns.

optimization

once a hit is generated through library screening, optimization of the

compound of interest takes place. This step involves multiple repeti-

tions of chemical modification of the hit to develop compounds with

desired properties. chemists need to know as soon as possible that

these reactions are proceeding as desired.

Samples were analyzed on an acQUiTY UpLc System with a Sample

organizer. The column was an acQUiTY UpLc BeH c18 (1.7 µm,

2.1 x 50 mm) run at 30 °c.

The injection volume was 5 µL. compounds were separated using a

generic water/acetonitrile gradient that was 1.1 min long.

detection was done with an acQUiTY UpLc pda, acQUiTY UpLc

eLS and an SQ Mass detector with an eSci source for eSi/apci

switching. Single samples were logged into the system using

openLynx open access and processed with the openLynx

application Manager.

Figure 5. Chromatograms from various times during a 60-minute reaction.

during the compound optimization stage of a discovery cycle,

medicinal chemists are not only interested in determining the key

structural features responsible for activity and selectivity, but also

what structural changes need be made to improve these characteris-

tics. Because the reactions necessary to bring about these changes

may take a long time, chemists need to be sure they are progressing

as expected.

Page 8: Waters Medicinal Chemistry Applications Book · 3 The Role of liquid ChRomaTogRaphy and mass speCTRomeTRy in mediCinal ChemisTRy “ Medicinal chemistry is a scientific discipline

Waters Corporation 34 Maple Street Milford, MA 01757 U.S.A. T: 1 508 478 2000 F: 1 508 872 1990 www.waters.com

Waters, acQUiTY UpLc, UpLc, and eSci are registered trademarks of Waters corporation. autopurification, autopurify, FractionLynx, i-FiT, LcT premier, LockSpray, MassLynx, odB, openLynx, profileLynx, Quattro premier, Quanoptimize, XBridge, and The Science of What’s possible are trademarks of Waters corporation. all other trademarks are the property of their respective owners.

©2007 Waters corporation. printed in the U.S.a.June 2007 720002099eN LB-kp

By using a walk-up UpLc/MS system, chemists were able to quickly

and easily monitor their reactions, noting the relative amounts of

starting materials and products. They were also able to note the

formation of any side products and make the necessary alterations to

minimize these in their reaction protocol.

ConClusion

We were able to increase throughput and data quality by

combining UpLc with a variety of detection techniques and

software solutions.

n screening: By combining the speed of the acQUiTY UpLc

System with the capacity of the Sample organizer, we were able

to nearly quadruple the screening throughput of the lab, without

sacrificing data quality.

n Confirmation: With the open access interface, medicinal

chemists were able to confirm the elemental composition of

their compounds, with minimal instrument training. The i-FiT

algorithm simplified the final exact mass determination by

reducing the number of possible elemental formulas.

n purification: We were able to use analytical Lc/MS data to tai-

lor the purification method to maintain the analytical resolution.

n Compounds profiling: The determination of physciochemical

properties was simplified with the use of the profileLynx

application Manager, which automated the calculations of

solubility, logp, metabolic stability, and cHi. The combination

of the column Manager and Quanoptimize facilitated the

development of optimal MS/MS method.

n optimization: chemists were able to quickly and easily log in

their samples to determine the progress of the reaction. They

were able to see the results of the analyses within minutes.

Page 9: Waters Medicinal Chemistry Applications Book · 3 The Role of liquid ChRomaTogRaphy and mass speCTRomeTRy in mediCinal ChemisTRy “ Medicinal chemistry is a scientific discipline

SCREEN

ING

Page 10: Waters Medicinal Chemistry Applications Book · 3 The Role of liquid ChRomaTogRaphy and mass speCTRomeTRy in mediCinal ChemisTRy “ Medicinal chemistry is a scientific discipline

inT RoduCT ion

Many compound libraries contain compounds that were synthesized

several years prior or obtained from outside resources. it is important

that the expected composition of each compound be confirmed. Lc/

MS has become the standard technique for confirming the purity and

identification of a compound that has demonstrated activity in a

biological screen.

if the library store is not routinely checked, false positives in an

activity screen are highly possible. This will lead to wasted time,

effort, and money on compounds that should not advance in the

discovery process. Because these libraries may contain thousands, if

not millions, of compounds, an open access Ultraperformance Lc®

(UpLc®)/MS system was investigated for high-throughput library

quality control.

enhancements to HpLc and Lc/MS technologies have provided use-

ful tools to improve the throughput and accuracy of these assays.

Throughput can be substantially increased with the use of UpLc/

MS, which makes use of small column particles (sub-2 μm) and

high operating pressure (>10,000 psi). This can result in an up to

10-fold increase in throughput along with a three-fold increase in

sensitivity.

due to the large number of samples analyzed and data generated

during this testing, a new software package has been created that

facilitates administration of this open access system. it created new

project directories for the users and moved the resulting project data

(such as raw data files) across the network as it was created. data

processing could then be done on a separate dedicated computer.

The software also monitored the instrument pc, providing on-the-fly

information about its status and the status of its sample queue from

a centralized location.

S YS T EM MA NAG EM EN T T OO L S FO R A H IG H -T H ROUG H P U T O P EN AC C E S S U P L C / M S S YS T EM US E D DU R ING T H E A NA LYSIS O F T HOUSA N DS O F SAM P L E S

darcy Shave, Warren potts, Michael Jones, paul Lefebvre, and Rob plumb Waters corporation, Milford, Ma, U.S.

eX peRimenTal

all experiments were conducted using the Waters® ZQ™ Mass

detector, equipped with an acQUiTY UpLc® System with a Sample

organizer, photodiode array (pda) detector, cooled autosampler

and column Heater. The ZQ was equipped with an eSci® source,

running in the eS+ ion mode. The instrumentation was controlled

by MassLynx™ 4.1 Software with openLynx™ and openLynx open

access application Managers.

Samples were run on a 1 min gradient from 5 to 95% organic at 0.8

mL/min. The column was a 1.7 µm, 2.1 x 50 mm acQUiTY UpLc BeH

c18 column. The pda was set to analyze a wavelength range from

210 to 400 nm. The mass detector analyzed a mass range from

100 to 500 amu with a dwell time of 100 ms and an interscan

delay of 50 ms.

eight microtitre palates, each containing 96 pharmaceutical samples,

were logged onto the system using openLynx open access. The first

and last samples in each plate were used for quality control.

The ACQUITY UPLC System with the ZQ Mass Detector for open access laboratories.

Page 11: Waters Medicinal Chemistry Applications Book · 3 The Role of liquid ChRomaTogRaphy and mass speCTRomeTRy in mediCinal ChemisTRy “ Medicinal chemistry is a scientific discipline

12

ResulTs and disCussion

By using an acQUiTY UpLc System with the optional Sample

organizer, we were able to analyze 3840 samples in under seven

working days on a single column. on a traditional HpLc system,

this would take approximately 27 working days, assuming a

10-minute run time.

The open access interface allowed users to log in the samples while

providing a minimal amount of information. a series of methods,

each including gradient conditions, MS conditions, and processing

parameters, was designed by the system administrator. The users

simply chose a method from this list, imported their sample lists, and

placed their microtitre plates in the indicated positions.

The samples were then analyzed and the data was processed. once

processing was finished, the data was copied to a file storage pc.

From here the users could do further processing if desired. as well,

a report file was generated from the processed file and converted to

.xml format. This facilitated storage of the results in a database.

instrumentation

Throughput was increased by using UpLc. This technique made use

of 1.7 μm column particles and high operating pressure (12,000

psi). These properties resulted in a five-fold increase in throughput.

Sensitivity was not investigated.

due to the large number of samples being run, an acQUiTY UpLc

Sample organizer was also used. This thermally-conditioned sample

storage compartment extended the capacity of the system by adding

space for seven deep-well microtitre plates (or 21 shallow-well plates).

Total sample capacity was increased from 192 samples (two plates)

to 768 samples (eight plates) when using 96-well plates. if using

384-well plates, maximum capacity would be 8064 samples.

an added advantage of the Sample organizer in an open access

environment is the ability to add samples to the system without

pausing the sample queue. When the door to the Sample Manager

is opened, any movement – whether of the sample plate or of the

needle – is paused for safety. This pause does not occur when loading

the Sample organizer.

software administration tools

The open access software allowed chemists to walk up to a terminal

and log in samples onto an instrument, inputting the minimum of

information needed for the sample run. it also allowed the system

administrator to maintain control over the open access systems and

to track the performance of each system. it facilitated batch process-

ing and reporting of results.

The administrator selected the fields that appeared when remote users

logged in samples. The administrator designated fields as manda-

tory so that login would not proceed unless the remote users entered

values for these fields. They also defined upper and lower limits for

the values of numeric fields. in addition, the administrator defined

the format for text that remote users entered in the text fields.

The open access Toolkit (oaToolkit) service ran on the acquisition

pc and copied open access users’ batch files and raw data to remote

locations once their samples were run. The information about these

users, and the locations to where their data was to be sent, is con-

tained within the administration tool. This information is uploaded to

the service on the acquisition pc.

The illustration in Figure 1 and following procedure describe the

order of events during typical operation.

1. The administrator uses the administration Tool to create a user.

2. The administrator uses the administration Tool to add extra

information about the oaLogin user, for example, that the raw

data of any of the user’s samples should be moved to the File

Storage pc whenever a user’s sample is processed.

3. The administrator uploads the user information to the oaLogin

pc. This adds the user’s name to the drop-down list in the

login screen on the oaLogin pc.

4. The administrator uploads the user information to the oaToolkit

service on the acquisition pc. The service now contains the

instructions of how to proceed if the oaLogin user logs in a batch.

5. The oaLogin user logs in a sample using the oaLogin terminal

as normal.

6. oaLogin logs the sample with MassLynx.

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13

7. When MassLynx has finished running the sample, the oaToolkit

service reads the batch file (.olb), and registers that it is from

a recognized user.

8. The oaToolkit service moves the raw information to the

specified location on the File Storage pc.

Reporting

The open access software allowed the administrator to define how

samples were processed. once all the data for a sample set had

been collected, the openLynx application Manager automatically

processed the data and created an openLynx Browser report (.rpt).

The browser report (Figure 2) presented a summary of results as a

color-coded map (found/not found/tentative) for easy visualiza-

tion of analysis results. Users accessed and reviewed the data by

simply pointing and clicking on the sample location of interest.

chromatograms, spectra, sample purity, peak height, peak area,

retention time, and other information can easily be reviewed within

the browser.

Figure 1. Data from the mass spectrometer is captured by the Acquisition PC, then is managed by the system administrator or accessed by the individual user via the OALogin tool. The raw data is also backed up to a File Storage PC.

Mass Spectrometer

Acquisition PC

OAToolkitAdministration PC

OAToolkitAdministration PC

File Storage PC

1.

2.3.

4.

5.

6.

7. 8.

The browser report was created in the report folder of the current

project. a secondary report location could have been specified, but

was not. The toolkit service also allowed for a copy of the report to be

sent over the network to another location. That location was specific

to each user – a folder on their office pcs. The users no longer had to

access the acquisition pc to view their reports. in addition, the raw

data folders were moved across the network to each user’s pc and

the users were able to reprocess it with a process-only version of

MassLynx.

Finally, the oaToolkit service was used to automatically convert

the browser reports to .xml format. This was accomplished using the

included .xml import and .xsl export schema. This data can then be

easily incorporated into a database or shared with colleagues.

system monitoring

on the administration pc, the Remote Status Monitor (RSM) moni-

tored the status of the open access acquisition pc, along with other

acquisition pcs on the network and wrote that monitoring information

to an .xml file. The information could then be read and interrogated

remotely in a browser (Figure 3).

Figure 2. OpenLynx browser report.

Page 13: Waters Medicinal Chemistry Applications Book · 3 The Role of liquid ChRomaTogRaphy and mass speCTRomeTRy in mediCinal ChemisTRy “ Medicinal chemistry is a scientific discipline

Waters Corporation 34 Maple Street Milford, MA 01757 U.S.A. T: 1 508 478 2000 F: 1 508 872 1990 www.waters.com

More detailed information about an instrument can be displayed

by clicking anywhere in the instrument row (Figure 4).

Figure 3. Status of the Open Access Acquisition PC.

ConClusion

Waters open access systems give chemists the ability to analyze

their own samples close to the point of production by simply walk-

ing up to the Lc/MS system, logging in their samples, placing their

samples in the system as instructed, and walking away. as soon as

the analysis is completed, sample results are emailed or printed

as desired. System configuration and setup is enabled through a

system administrator who determines login access, method selec-

tion, and report generation.

openLynx oaToolkit enables administrators to manage open

access users from a central point, assigning detailed configuration

information and attributes for these users, and then exporting these

details to multiple oaLogin pcs and acquisition pcs. openLynx

oaToolkit also enables administrators and users to remotely moni-

tor the status of acquisition pcs.

Figure 4. Detailed view of instrument status.

Waters, acQUiTY UpLc, eSci, Ultraperformance Lc, and UpLc are registered trademarks of Waters corporation. MassLynx, openLynx, ZQ, and The Science of What’s possible are trademarks of Waters corporation. all other trademarks are the property of their respective owners.

©2006-2007 Waters corporation. printed in the U.S.a.June 2007 720001482eN LB-kp

Page 14: Waters Medicinal Chemistry Applications Book · 3 The Role of liquid ChRomaTogRaphy and mass speCTRomeTRy in mediCinal ChemisTRy “ Medicinal chemistry is a scientific discipline

oV eRV ieW

Maximum efficiency is essential for Lc/MS labs challenged by

throughput requirements and the management of data from a vari-

ety of systems and users. analyzing routine samples and returning

the results to chemists can easily consume an analyst’s entire

day, leaving them with little time to focus on tasks that require

their expert attention. Walk-up open access systems allow chem-

ists to analyze their own samples, freeing up analysts’ time for

more challenging analyses without compromising the quality of the

final results.

The Waters® openLynx™ open access application Manager for

MassLynx™ Software offers the power of chromatography and mass

spectrometry to chemists who are not analytical instrumentation

specialists. To minimize the learning curve for instrument operation,

openLynx open access leads chemists through sample submission,

method selection, and reporting options. The system is maintained

by a system administrator who predefines the system configuration,

available experimental methods, processing criteria, and reporting

options. By allowing chemists to submit their own samples, routine

analyses can be performed more efficiently, leaving instrumentation

experts more time to focus on advanced analyses.

inT RoduCT ion

open access lC/uV, lC/ms, lC/ms/ms, and gC/ms

The openLynx open access application Manager is designed to allow

chemists to walk up to a terminal and log in samples onto an instru-

ment, while inputting the minimum of information needed for the

sample run. openLynx open access allows the system administrator

to maintain control over the open access systems and to track the

performance of each system. it also facilitates batch processing and

reporting of results.

O P EN LYN X O P EN AC C E S S

openLynx open access offers comprehensive capabilities:

n simplified sample submission process – a single page login

or a step-by-step, wizard-enabled process allows users to enter

their name and sample information, and select pre-determined

experimental methods and processing criteria

n exact mass measurement utilization – For use with the

appropriate mass spectrometers

n summary report generation – Reports are automatically

printed, emailed, and viewed via the openLynx browser,

containing sample found/not found information, purity,

probable elemental composition (with exact mass MS),

chromatograms, and spectra

n Walk-up optimization of ms/ms methods and quantification

of compounds of interest – combines openLynx open access

with Quanoptimize™ and QuanLynx™ application Managers

n advanced search – Spectral library generation and searching

n automation of routine system administration tasks –

Through the use of openLynx open access Toolkit (oaToolkit)

sof T WaRe seTup

defining parameters

openLynx open access allows remote users to run samples on

the acquisition computer. For openLynx open access users to be

successful, the administrator defines (via the openLynx method)

the sample information that users must provide when running

samples. an intuitive oaLogin setup wizard simplifies the system

configuration and administration workspace to include only the ana-

lytical features the administrator uses.

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The administrator selects the fields that appear when remote users log

in samples using openLynx open access via the Walk-up tab of the

openLynx method (Figure 1). They can designate fields as mandatory

so that login will not proceed unless the remote users enter values for

these fields. They can also define upper and lower limits for the value

of numeric fields. in addition, the administrator can define the format

for text that remote users enter in text fields.

setting options for users

Using the administrator mode of openLynx open access, the admin-

istrator defines how users login samples via a number of options

(Figure 2). Login setup ranges from changing the window appear-

ance to allowing users to create their own user name. Notification of

users via email can be enabled, as can barcode support. oaLogin can

be configured for use with either openLynx (sample processing) or

autopurify™ (fraction processing).

Figure 1. OpenLynx method showing some of the OpenLynx Open Access input fields. Figure 2. Administrator-set OpenLynx Open Access options.

setting file options

The administrator sets several file options. These include specifying

the location where the openLynx methods, openLynx status file, and

HpLc files are located. The administrator can set which methods are

visible to users, along with the format needed for the text fields.

Configuring quality control runs

The administrator can configure openLynx to check that the Lc and

MS instrumentation are working correctly, thus ensuring the consis-

tency of the data. The quality control feature (Figure 3) allows users

to run a standard and have it compared to the results of the same

standard that was run at an earlier time. Values that can be used

to confirm system operational performance include peak retention

time, peak area, the presence of specific masses or wavelengths, and

spectral intensity.

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Before a Qc comparison can be run to check the system, there must

be an openLynx method that contains the expected results from a

standard. The Qc run acquires data from a sample with a known

retention time and peak intensity and then compares the results to

the values defined in the openLynx method.

openlynx open access Toolkit (oaToolkit)

openLynx oaToolkit allows the creation and administration of

openLynx open access users. it can push user information to

openLynx open access pcs across the same network, as well as

gather existing openLynx open access user information from

openLynx open access pcs. it can create new project directories for

the openLynx open access users and can move the resulting project

data (such as raw data files) as it is created. The software can monitor

numerous instrument pcs, providing on-the-fly information about

their status as well as the status of their batch queues – all from

a central location. it ensures confidence in analytical results with

password protection for open access users.

Figure 3. OpenLynx Open Access quality control options.

The openLynx oaToolkit includes the following key features:

n administration Tool (Figure 4) – enables an administrator to

create and manage all openLynx open access users from a

single pc, and replicates that information to multiple openLynx

open access pcs and acquisition pcs

n oaToolkit service – Runs in the background on one or more

acquisition pcs, monitors sample batches submitted by

openLynx open access users that were uploaded from the

administration Tool

n Remote status monitor (Figure 5) – enables any user to

monitor the status of acquisition pcs and their batch queues

from a single pc

Figure 4. OpenLynx Toolkit Administrator Tool.

Figure 5. Remote Status Monitor.

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additionally, the openLynx oaToolkit Service:

n Relocates data produced during the processing of an openLynx

open access user’s batch of samples

n creates new project folders in which to store the processing

data on a timed basis

n converts report files to different formats (XML, HTML, or text)

logging samples

login samples window

Running samples using openLynx open access (Figure 6) involves

entering sample information to correctly identify the samples and

loading the samples into the autosampler. The methods available to

the users depend on selections made by the administrator.

if the administrator enables user passwords (using openLynx

oaToolkit), the user must enter their designated password before they

can login samples (Figure 7). if they enter an incorrect password, an

error message appears and they cannot continue until the correct

password has been entered.

single-page log-in vs. wizard

openLynx open access displays the wizard for sample login by

default. However, the administrator can allow openLynx open access

users to use a single-page dialog box (Figure 8) for “single shot”

samples. Users can enter multiple samples in this way. openLynx

open access views the samples logged in as a single job.

Figure 6. OpenLynx Open Access window.

Figure 7. Entering user password.

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The single-page login contains most of the selections on the

wizard pages (Figure 9) necessary to schedule samples. The benefit

of the single-page login is the speed of entering information for a

single sample in a single dialog box, rather than through a wizard.

This wizard is beneficial when logging in larger sample sets.

loading samples into the autosampler

There are two ways to load samples into the autosampler. The

system administrator designates each plate in the autosampler

as either “single shot” or “whole plate” login. if a plate is desig-

nated for single shot login, the user enters data for their samples

manually or imports data from a tab-delimited text file. openLynx

assigns available positions for the samples on existing plates. if a

plate is designated for whole plate login, the user prepares data in

a spreadsheet or as a text file and imports it into openLynx open

access. This is useful if the user needs to run a large number of

samples in one run. openLynx reserves the entire plate for samples

and the user selects the sample locations.

Typically, a system with multiple plates will have both single shot and

whole plate login available.

Figure 9. With the wizard, walk-up users enter their name, choose a method, enter sample information, and place the sample in the autosampler.

p RoC essing samples

processing data automatically

The administrator determines how openLynx processes the open

access results. To configure openLynx open access to process data

automatically, the administrator must create an openLynx method

that defines the processing parameters.

The administrator must define the integration parameters for the type

of data they want to process:

n ms+ data – For positive ions (total ion chromatogram (Tic),

base peak intensity (Bpi), and mass chromatograms)

n ms– data – For negative ions (Tic, Bpi, and mass chromatograms)

n analog data – For up to four channels of analog chromatograms

n dad data – For total absorbance chromatogram (Tac), Bpi,

and wavelength chromatograms

Specifying how peak detection occurs involves selecting the

integration algorithm and parameters that control peak detec-

tion, enabling smoothing (if desired), and setting the smoothing

parameters and setting threshold values.

Figure 10. Chromatogram integration window.

When setting the integration and peak detection parameters (Figure

10), the administrator can specify which integration algorithm

(standard or apexTrack™) to use; how the baseline will be treated

for valleys, peak tailing, and drift; and how peak separation for fused

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peaks and shoulders will be handled. By enabling smoothing,

noise will be decreased by filtering data points. Smoothing types

include Savitzky-Golay and mean. The threshold values are set

for one or more of the four threshold parameters: relative and

absolute height and relative and absolute area. This option is

used to remove peaks whose height or area is less than a specified

percentage of the highest peak.

in addition to acquiring and processing data, quantitation and optimi-

zation can be performed through openLynx open access.

performing quantitation

open access quantitation is a way for the user to run quantitation

analysis through openLynx open access (Figure 11). openLynx

stores the conditions required for a particular quantitation analysis

in an openLynx method. openLynx open access users select the

openLynx method during login.

Figure 11. Open Access quantitation parameters.

Using open access quantitation, openLynx open access users

can quantify the results as data are acquired. The processing steps

available include:

n integrating samples

n Quantitating samples

n calibrating standards

using quanoptimize with openlynx open access

The optional Quanoptimize optimizes the acquisition and quantitation

parameters for a particular experiment. open access Quanoptimize

(Figure 12) generates MS and MS/MS parameters by optimizing

the cone voltage, parent ion, and collision energy parameters.

Quanoptimize then takes these MS methods and performs automated

acquisition and processing using processing methods developed on

the fly. it can quantify these results using specified methods. This

technique is useful for high throughput screening.

Figure 12. Open Access QuanOptimize parameters.

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RepoRT ing

Results reporting

Reporting in open access systems is facilitated by the openLynx

application Manager. openLynx can report results using a flexible

array of printed reports or through a results browser.

The standalone openLynx browser (Figure 13) is an interactive

tool for viewing openLynx results and can be run on any windows

pc without requiring a full MassLynx installation. chemists can

use the browser on their desktop pc to view the results (.rpt file

format) that had been automatically emailed to them at the end of

openLynx processing.

The openLynx browser presents a summary of results as a color-

coded (found/not found/tentative) map for easy visualization of

analysis results. chemists can access and review the data supporting

any found/not found/tentative assignment by simply pointing and

clicking on the sample location of interest. chromatograms, spectra,

sample purity, peak height, peak area, retention time, and other

information can easily be reviewed within the browser.

Figure 13. OpenLynx browser.

Page 21: Waters Medicinal Chemistry Applications Book · 3 The Role of liquid ChRomaTogRaphy and mass speCTRomeTRy in mediCinal ChemisTRy “ Medicinal chemistry is a scientific discipline

Waters Corporation 34 Maple Street Milford, MA 01757 U.S.A. T: 1 508 478 2000 F: 1 508 872 1990 www.waters.com

printing and distributing reports

openLynx creates an openLynx browser report file (.rpt) after

it finishes a run and processes the data. This file resides in the

openLynx open access\Reportdb folder. The file is named with the

job number followed by the extension .rpt when the user logs in to

openLynx. openLynx report files may be exported in .txt, .tab, .csv,

and .xml formats.

The administrator can configure openLynx open access so remote

users can find the reports that openLynx generates after running

samples. information such as where to store reports and what print

report format to use can be specified.

Waters is a registered trademark of Waters corporation. MassLynx, Quanoptimize, QuanLynx, apexTrack, autopurify, openLynx, and The Science of What’s possible are trademarks of Waters corporation. all other trademarks are the property of their respective owners.

©2006-2007 Waters corporation. printed in the U.S.a.June 2007 720001594eN LB-kp

ConClusion

The openLynx open access application Manager provides

comprehensive, easy, and flexible open access walk-up Lc/UV,

Lc/MS, Lc/MS/MS, and Gc/MS systems operation management

for laboratories that have chemists with varying levels of instru-

mental analysis experience. With customizable batch processing

and results review to support the large amounts of data resulting

from high throughput analyses, a highly productive environment is

ensured for high-volume laboratories.

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CoNfIR

matIo

N

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inT RoduCT ion

The identity and purity of a candidate pharmaceutical is critical to

the effectiveness of the drug screening process. Lc/MS is employed

extensively in drug discovery in order to exclude false positives and

maintain the high quality of the product. This process can be time

consuming and can potentially delay the progression of a drug

through the discovery process.

Thus, sample throughput is a critical issue in moving compounds

from the hit to lead status. Ultraperformance Lc® (UpLc®) lever-

ages sub-2 µm Lc particle technology to generate high efficiency

faster separations.

When a photodiode array/evaporative light scattering/mass spec-

trometry (pda/eLS/MS) detection scheme is used in conjunction

with multiple-mode ionization, the potential for peak detection is

greatly improved. pharmaceutical chemical libraries often contain

a great diversity of small molecules to cover a broad range of

biological targets.1 in this environment, the ability to obtain infor-

mation pertaining to multiple MS acquisition modes, in addition to

pda and eLS, in a single injection is invaluable.

open access software offers the power of chromatography and

mass spectrometry to chemists who are not analytical instru-

mentation specialists. it allows them to quickly and easily know

what they’ve made and allows the experts to work on the difficult

analytical problems.

an open access UpLc/MS system was investigated for high

throughput library Qc. in this application note, we describe some

of the enhancements to Lc and Lc/MS technologies that have

generated useful tools that improve the throughput and accuracy

of these assays.

Figure 1. The ACQUITY SQD for open access.

N E W T OO L S FO R IM P ROV ING DATA QUA L IT Y A N D A NA LYSIS T IM E FO R C H EM IC A L L IB R A RY IN T EG R IT Y A S S E S SM EN T

Marian Twohig, paul Lefebvre, darcy Shave, Warren potts, and Rob plumb Waters corporation, Milford, Ma, U.S.

eX peRimenTal

lC conditions

Lc system: Waters® acQUiTY UpLc® System

column: acQUiTY UpLc BeH c18 column

2.1 x 30 mm, 1.7 µm

column temp.: 50 °c

Sample temp.: 8 °c

injection volume: 2 µL

Flow rate: 800 µL/min

Mobile phase a: 0.1% Formic acid in water

Mobile phase B: 0.1% Formic acid in acetonitrile

Gradient: 5 to 95% B/0.70 min

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24

ms conditions

MS system: Waters SQ detector

ionization mode: eSi positive/eSi negative,

multi-mode ionization

capillary voltage: 3.0 kV

cone voltage: 20 V

desolvation temp.: 450 °c

desolvation gas: 800 L/Hr

Source temp.: 150 °c

acquisition range: 100 to 1300 m/z

Scan speed: 2500, 5000, and 10,000 amu/sec

Note: A low volume micro-tee was used to split the flow to the ELS and SQ.

els conditions

Gain: 500

N2 gas pressure: 50 psi

drift tube temp.: 50 psi

Sampling rate: 20 points/sec

pda conditions

Range: 210 to 400 nm

Sampling rate: 20 points/sec

ResulTs and disCussion

Maximum efficiency is essential for labs challenged by throughput

requirements and the management of data from multiple systems

and users. The Waters open access suite of software streamlines

the integration of analysis with data acquisition, processing, and

reporting.

The system and software are initially configured by a system admin-

istrator who defines login access, method selection, and reporting

schemes. This allows users to analyze their own samples with mini-

mal intervention from analytical support.

sample login

openLynx™ open access application Manager for MassLynx™

Software is designed to allow chemists to walk up to a terminal and

log in samples while entering the minimum information required to

run the samples. a series of methods, each including gradient and

MS conditions as well as processing parameters, are initially set up

by the system administrator. The users choose an appropriate method

from the list, importing their sample lists and placing their samples

in the position designated by the software. desired sample analysis

is then performed by the configured system. The single page login

window can be seen in Figure 2.

open access system

chromatographic separations were carried out using the acQUiTY®

SQd System coupled to detectors specialized for UpLc separations:

the single quadrupole SQ Mass detector, and pda and eLS detectors

that provided simultaneous signal collection. For additional flexibil-

ity, the UpLc system was configured with a Sample organizer and a

column Manager. The sample capacity of the system totals twenty two

384-well plates, for 8448 library samples in total. This extends the

overall walk-away time for the system. The column manager allows

four UpLc columns to be installed, heated, and switched into line

Figure 2. OpenLynx Open Access single page login.

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25

Figure 3. Chromatograms shown at 2500, 5000, and 10,000 amu/sec.

Time0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70

%

0

100

0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70

%

0

100

0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70

%

0

0.30

0.26

0.53

0.30

0.260.53

0.30

0.250.53

2500 amu/sec

5000 amu/sec

10,000 amu/sec

Figure 4. Spectrum for isotope model and for acquired spectrum.

m/z305 310 315 320 325 330

%

0

100

%

0

100 319

321

319

321

C17H20N2CISAcquired Spectrum10,000 amu/sec

C17H20N2CISIsotope Model

based on the method requirements. This allows the chemist to take

advantage of the broad range of stationary phases that encompass

compound types, ranging from very hydrophilic to very lipophilic.

sample analysis

Samples were analyzed using gradients less than one minute in

length with a flow rate of 800 µL/min. When analyzing the narrow

peaks generated by the UpLc/MS system, the data collection rate can

compromise the number of points across the Lc peak, resulting in a

poor definition of the eluting peak and hence inaccurate results.

The ability of the MS system to collect data at a high scan speed,

10,000 amu/sec, greatly improves chromatographic peak defini-

tion. This in turn facilitates the acquisition of a large number of

individual acquisition modes in one run while maintaining adequate

peak characterization.

as can be seen from the data displayed in Figure 3, the result of

operating at lower data collection rates can compromise the chro-

matographic resolution. To maintain chromatographic integrity, it is

therefore advantageous to be able to scan at elevated scan speeds.

The total cycle time of the method was 1 minute 20 seconds,

facilitating increased sample throughput while still allowing gener-

ous washing steps to prevent sample-to-sample memory effects.

Using a flow rate of 800 µL/min and a 2.1 x 30 mm column clears

9 column volumes/min.

The spectral data quality of scanning experiments carried out from

2500 to 10,000 amu/sec were found to be comparable, thus provid-

ing confidence that operating at these rapid data collection rates

does not compromise the spectral data quality. Figure 4 shows a

comparison of an acquired spectrum with a software generated isoto-

pic model. isotope ratios of data collected at 10,000 amu/sec were

within 1% of the isotopic model, again ensuring data fidelity is not

compromised.

in addition to obtaining mass confirmation by multiple MS modes, it

is possible to add pda and eLS detectors to obtain auxiliary informa-

tion. a single run can then provide UV spectral information and an

estimation of compound purity at low wavelengths.

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26

-

-

Time0.10 0.20 0.30 0.40 0.50 0.60 0.70

%

0

100

0.10 0.20 0.30 0.40 0.50 0.60 0.70

%

16

0.10 0.20 0.30 0.40 0.50 0.60 0.70%

12

0.10 0.20 0.30 0.40 0.50 0.60 0.70

%

0

100

0.10 0.20 0.30 0.40 0.50 0.60 0.70

LS

U

0.00020.00040.000

0.10 0.20 0.30 0.40 0.50 0.60 0.70

AU

0.01.0e-1

0.31

0.300.24 0.54

0.310.54

0.640.180.10

0.560.24 0.32 0.41 0.48

0.54

0.310.25 0.54

PDA

ELS

APcI+

APcI-

ESI-

ESI+

Figure 5. UPLC/PDA/ELS/MS with multi-mode ionization.

Figure 6. The OpenLynx browser.

eLS detection is an alternative to UV detection, and does not depend

on the presence of a chromaphore. eLS detection works by measur-

ing the light scattered from the solid solute particles remaining after

nebulization and evaporation of the mobile phase. chromatograms

illustrating the use of triple detection (pda/eLS/MS) are shown in

Figure 5. The signal from an eLS detector can give a tentative estima-

tion on the relative quantities of the components present. it has been

known to give rise to similar responses for related compounds.2

The chromatographic peak widths of the MS and eLS increased by

25 to 30% when compared with the pda trace. This can be attributed

to the use of a low volume micro-tee after the pda.

data processing

as soon as the analysis is complete, data is automatically pro-

cessed and a sample report is generated. Reporting in open access

systems is facilitated by the openLynx application Manager.

openLynx can report results using printed reports or through the

openLynx browser. The browser presents a summary of the results

as a color coded (found/not found/tentative) map for clear interpre-

tation of the results. chromatograms, spectra, sample purity, peak

height, peak area, retention time, and other information can easily

be viewed by the browser. The openLynx browser, shown in Figure

6, displays the results for the entire 384-well plate. The report can

automatically be emailed, converted to pdf, or printed as desired.3

The openLynx oaToolkit facilitates an even easier administra-

tion of an open access system, automating many of the system

management tasks carried out by a system administrator. The

software also remotely monitors the status (via the Remote

Status Monitor module) of one or more acquisition pcs and writes

monitoring information to an XML file. The status summary page

opens in the browser and contains a list of acquisition pcs, and

the number of samples pending in the queue.4 This allows the

chemist to select the instrument with the shortest wait time, again

increasing productivity.

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Waters Corporation 34 Maple Street Milford, MA 01757 U.S.A. T: 1 508 478 2000 F: 1 508 872 1990 www.waters.com

ConClusion

it is important to verify the identity and purity of a compound before

early activity studies. chemists need to be sure they have synthesized

the expected compound. Because large numbers of compounds may

be created, it is necessary for this screening to be high throughput.

and because only a small amount of material is synthesized, the

screening must also consume as little material as possible, while

generating a diverse amount of information.

The described system and software combination can autonomously

evaluate large numbers of samples with a cycle time of 1 minute and

20 seconds. data can then be automatically processed and a sum-

mary report can be generated. The scan speed capabilities of Waters

acQUiTY SQd System make it possible to better characterize narrow

chromatographic peaks. This has become a necessity since the advent

of sub-2 µm particle technology where chromatographic peaks can be

1 second wide or less.

Signals from auxiliary detectors such as pda and eLS can be col-

lected simultaneously. Together with the MS data, they provide

important information relating to purity and an estimation of the

relative quantities of the components present.

open access gives the chemist a walk-up system that is flexible for

analytical data acquisition. it runs as a complete system, from sample

introduction to end results.

The use of the fast-scanning MS along with the throughput of UpLc

technology and remote status monitor software allows the chemist to

obtain high quality comprehensive data about their compounds in the

shortest possible time. This combined with intelligent open access

software allows informed decisions to be made faster, thus support-

ing the needs of the modern drug discovery process.

References

1. Mike S. Lee, Lc/MS applications in drug development, Wiley-interscience Series on Mass Spectrometry. 2002; (chapter 6) 96-106.

2. kibbey, c.e. Mol. diversity. 1995; i: 247-258.

3. darcy Shave, openLynx open access, Waters application Library. 2006: 720001594eN.

4. darcy Shave, openLynx oaToolkit for open access Systems, Waters application Library. 2006: 720001469eN.

Waters, acQUiTY, acQUiTY UpLc, Ultraperformance Lc, and UpLc are registered trademarks of Waters corporation. MassLynx, openLynx, and The Science of What’s possible are trademarks of Waters corporation. all other trademarks are the property of their respective owners.

©2007 Waters corporation. printed in the U.S.a.June 2007 720002257eN LB-kp

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puRIfICa

tIoN

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inT RoduCT ion

purification laboratories face many of the same challenges that

their counterparts in analytical laboratories face: the need to

increase throughput and efficiency without sacrificing quality and

quantity. Successful performance of a purification lab is measured

in the ability to produce pure fractions in sufficient quantities in a

timely manner.

Ultraperformance Lc® (UpLc®) has been widely accepted by

chromatographers because of the improvements over HpLc in

sensitivity, resolution, and speed of separations. Now scientists

are beginning to explore the use of this technology in the sample

screening process as a screening tool to evaluate samples prior

to purification.

a typical run time for analytical screening in a preparative lab is

10 minutes. By capitalizing on the efficiency of UpLc, a 10-minute

run time can be shortened to as little as 1 minute. This offers sub-

stantial time savings enabling for greater capacity, but also fits

into the “fail fast and fail cheap” motto adopted by many pharma-

ceutical companies.

This application note will discuss the use of focused gradients to

maintain selectivity and resolution and to allow UpLc screening

to be applied to preparative samples. This will offer the substantial

time savings associated with UpLc to customers in the preparative

environment.

eX peRimenTal

a standard solution of pharmaceutical-like compounds was

prepared to simulate the conditions under which many purification

systems operate.

S C A L ING A S E PA R AT IO N F ROM U P L C T O P U R I F IC AT IO N US ING FO C US E D G R A DI EN T S

Ronan cleary, paul Lefebvre, and Marian Twohig Waters corporation, Milford, Ma, U.S.

uplC conditions

Lc system: Waters® acQUiTY UpLc® System with acQUiTY

UpLc photodiode array (pda) detector

column: acQUiTY UpLc BeH c18, 1.7 µm, 2.1 x 50 mm

injection volume: 2.0 µL

Flow rate: 0.8 mL/min, 2.1 x 50 mm

Mobile phase a: 0.05% Formic acid in acetonitrile

Mobile phase B: 0.05% Formic acid in water

Gradient: Generic 5 to 95% over 2 minutes

Focused Gradient

hplC conditions

Lc system: Waters autopurification™ System

column: Waters XBridge™ prep oBd™ c18,

5 µm, 19 x 50 mm

Waters XBridge c18, 5 µm, 4.6 x 50 mm

injection volume: 200 µL

Mobile phase a: 0.05% Formic acid in acetonitrile

Mobile phase B: 0.05% Formic acid in water

Figure 1. The mass-directed AutoPurification System.

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30

Flow rate: 22 mL/min

Gradient: 0 to 0.25 min, 2% B to initial % B

0.25 to 1.61 min, initial % B to end % B

1.61 to 1.86 min, end % B to 95% B

1.86 to 2.71 min, 95% B

2.71 to 2.72 min, 95% B to 2% B

ms conditions

MS system: Waters 3100 Mass detector

ionization mode: positive

Switching time: 0.05 sec

capillary voltage: 3 kv

cone voltage: 60 V

desolvation temp.: 350 °c

desolvation gas: 500 L/Hr

Source temp.: 300 °c

acquisition range: 150 to 700 amu

acquisition rate: 5000 amu/sec

ResulTs and disCussion

in order to maintain the selectivity and resolution achieved

by analytical analysis, the overall cycle time of a preparative

analysis must be increase almost nine fold.1 This long cycle time

is not practical for most separation scientists. Therefore, we look

to focused gradients to maintain selectivity and resolution in

UpLc screening.

The UpLc separation of the sample shows the compound of

interest eluting at 0.48 min, and is partially resolved from the

peak at 0.51 min.

The separation is first directly scaled to a 19 x 50 mm XBridge prep

oBd c18 column. The XBridge chemistry is built on the same second-

generation bridged ethyl hybrid (BeH) particle as the acQUiTY

UpLc BeH chemistry, in order to maintain the selectivity and

resolution of the analytical analysis. To maintain the resolution and

selectivity, the overall cycle time must be increased over nine fold.

Figure 2. ACQUITY UPLC analytical separation.

Time0.00 0.20 0.40 0.60

AU

0.0

1.0

2.0

0.480.27

0.130.23

0.34 0.51

Figure 3. Direct scale-up maintains resolution and selectivity, with a run time of 8 minutes.

Time2.00 4.00 6.00 8.00

AU

0.0

2.0e+1

4.0e+1 3.88

0.64

1.61

2.31 4.17

in a preparative environment, where the compound of interest is

being isolated from the other components in the sample, retaining

analytical resolution is not as important as isolating and collecting

the compound of interest.2

a set of focused gradients can be created based on the relationship

between percent composition and retention time. The system dwell

time is used to determine that relationship.3

Here, in the analytical screen the mobile phase is 2% organic solvent

at 0.17 minutes and 17.5% at 0.295 minutes, and so a series of

gradients can be created.

The theory behind the focused gradients is the same for HpLc

and for UpLc, but the time window for the UpLc gradient is

much smaller.

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31

method Time (min) Time (min) % B start % B end

a 0.17 0.295 2 17.5

B 0.295 0.42 17.5 33

c 0.42 0.545 33 48.5

d 0.545 0.67 48.5 64

e 0.67 0.795 64 79.5

F 0.795 0.92 79.5 95

Table 1. UPLC retention time windows and corresponding focused preparative gradient composition.

Based on Table 1, method c is selected to isolate the compound

that eluted at 0.48 min in the UpLc analysis. Using the focused

gradient, the separation and isolation of the compound was

carried out in 3 minutes.

Figure 4. Separation of the compound of interest using a 3-minute focused gradient.

Time1.00 2.00 3.00

AU

0.0

5.0e+1

1.0e+2

0.83

0.632.01

1.08 2.24

uplC library purity screening

This same methodology can be applied to the purity screening and

purification of a large sample library. The acQUiTY UpLc System’s

large capacity (22 384-well plates) and the rapid analysis cycle time

provide the ideal tool for high throughput library screening. data is

processed and handled using autopurify™, part of the FractionLynx™

application Manager™.4

focused library purification

autopurify automatically selects the samples requiring purification

and the corresponding focused preparative method.

Figure 5. AutoPurify processing report showing the color coded purity and found/not found of a 348-well plate.

Figure 6. AutoPurify processing of the UPLC screening library.

Page 32: Waters Medicinal Chemistry Applications Book · 3 The Role of liquid ChRomaTogRaphy and mass speCTRomeTRy in mediCinal ChemisTRy “ Medicinal chemistry is a scientific discipline

Waters Corporation 34 Maple Street Milford, MA 01757 U.S.A. T: 1 508 478 2000 F: 1 508 872 1990 www.waters.com

uplC fraction analysis

The substantial time savings associated with analytical screening can

be magnified by incorporating UpLc into the analysis of the collected

fractions. The collected fractions are analyzed to determine the new

sample purity, and sample lists are automatically generated for each

step of the process. By incorporating fraction analysis by UpLc into

the workflow, the efficiency of the lab is further increased.

ConClusionn Scale-up from UpLc to preparative HpLc in an efficient manner

is possible with the use of focused gradients.

n The efficiency of UpLc can be carried through to purification,

offering a substantial increase in throughput and productivity.

n The autopurify capabilities of FractionLynx allows for

automation from the initial UpLc Qc, through purification,

to UpLc fraction analysis.

n autopurify is also capable of automatically selecting a focused

preparative gradient based on the analytical results, giving

better quality purification and eliminating the need for expert

manual invention.

References

1. Xia F, cavanaugh J, diehl d, Wheat T. Seamless Method Transfer from UpLc Technology to preparative Lc, Waters application Note. 2007; 720002028eN.

2. cleary R, Lefebvre p. The impact of Focused Gradients on the purification process, Waters application Note. 2007; 720002284eN.

3. Jablonski J, Wheat T. optimized chromatography for Mass directed purification of peptides, Waters application Note. 2004; 720000920eN.

4. cleary R, Lefebvre p. purification Workflow Management, Waters application Note. 2006; 720001466eN.

Figure 7. AutoPurify processing of the UPLC analysis of the collect fractions.

Waters, acQUiTY UpLc, Ultraperformance Lc, and UpLc are registered trademarks of Waters corporation. autopurification, oBd, XBridge, autopurify, FractionLynx, application Manager, and The Science of What’s possible are trademarks of Waters corporation. all other trademarks are the property of their respective owners.

©2007 Waters corporation. printed in the U.S.a.June 2007 720002283eN LB-kp

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inT RoduCT ion

a standard requirement for drug discovery screening of synthetic

libraries is that the test compounds must have a minimum purity.

purity is based on the area percent of an Lc chromatogram from a

detector such as UV, evaporative light scattering (eLS), MS with

a total ion chromatogram (Tic), or a combination of multiple

detectors. if the screening compounds do not meet this standard,

purification is required. Managing the flow of samples, subsequent

fractions, and all the associated data through this process can often

be difficult and time consuming.

This application note illustrates how a sample is efficiently taken

through a three-step purification process utilizing the autopurify™

capabilities within the Waters® FractionLynx™ application Manager

for MassLynx™ Software, and the autopurification™ System for

MS-directed analysis. This comprehensive informatics solution

enables automation from the initial evaluation, through the purifica-

tion, to analysis of the collected fraction.

disCussion

The autopurify functionality uses the results of the analytical analy-

sis to determine the purification process. By performing an analytical

evaluation of the sample, the presence of the target compound is

confirmed and its purity measured (Figure 1).

P U R I F IC AT IO N W O R k F LOW MA NAG EM EN T

Ronan cleary and paul Lefebvre Waters corporation, Milford, Ma, U.S.

information determined from analysis of the fractions can be used

to help with post-purification handling such as fraction pooling and

transfer to an evaporator. a report can be exported in different file

formats such as .xml, .csv, and .tab, to easily interface with other

sample handling software packages.

The software will decide which shallow gradient should be used to

perform the purification (Figure 2).

Figure 1. TIC chromatogram of the analytical-scale analysis of the crude sample.

Then, it automatically performs analysis of the collected

fractions (Figure 3).

Figure 2. TIC chromatogram after purification, with fraction collection indicated by the shaded area.

Figure 3. TIC chromatogram of the analysis of the collected fraction.

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34

step 1: analytical interpretation

in the first of the three-step process, the purity of the target mass is

identified by integrating the chromatogram. in the example shown

in Figure 4, the area percent of the target determined from the Tic

(22%) is then used to calculate the sample purity.

The area percent can also be determined by total absorbance current,

wavelength, or analog signal. The purity of the target is then classi-

fied as “pass,” “tentative,” or “fail,” based on user-defined limits. in

this example, less than 10% pure means purification will not occur,

10 to 80% purity requires purification, greater than 80% is pure

enough, and does not require further purification.

in a manual process, the analyst would evaluate the separation, and

adjust the gradient to achieve the best results. However, in an open

access environment or where large numbers of samples are being

handled, automation is necessary.

step 2: The purification process

in the second step of the process, purification occurs. The software

will determine the purification method best suited to improving the

separation by choosing one of six different shallow gradients. Using

the analytical retention time of the target, the appropriate shallow

gradient-based method will be chosen.

Shallow gradients, also referred to as narrow gradients, allow

for optimal target separation from closely eluting impurities,

thus improving the purity of the resulting fraction. each narrow

gradient, whose time window is indicated by the colored lines

(Figure 5), is created to cover a different timed section of the

analytical gradient.

The analytical gradient is indicated by the dotted black line, and

shows the solvent change over the course of the gradient to be from 5

to 95% B. With the relationship between the analytical retention time

and the elution organic composition known, the software can choose

which of the narrow gradients will be used to automatically purify the

samples during the purification stage of the process.

When the software evaluates the analytical sample, it creates a

browser report defining the recommended strategy. The user has

the opportunity to change the strategy if necessary. The part of the

report that refers to the strategy is the results pane (Figure 6). in this

example, there are several other samples analyzed, but the one that

is of interest is that last one on the list, a123008.

Figure 4. Analytical evaluation of mass 357.1 is 22% of the TIC, and the target sample is co-eluting with peak 2. An overlay chromatogram of the two co-eluting peaks, with the spectrum, indicates the potential fraction contamination that could occur.

Figure 5. Graphical representation of analytical and prep gradients.

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35

The improved separation is more clearly displayed when the chro-

matograms of the two co-eluting compounds, as seen in Figure 4, are

extracted and their chromatograms reviewed. Figure 8 shows the two

chromatograms of masses 255 and 358, overlaid, and the improved

separation achieved.

The sample in this case eluted at 4.04 min (Figure 7), so the

narrow gradient chosen for the purification was “Narrow Gradient

c,” the one that targeted the solvent change that occurred between

4 and 5 minutes. This gradient is denoted by the green line, which

changes from 24 to 37% organic over 6.5 min, and is defined

graphically as below.

Figure 6. Browser results pane with sample purity and prep strategy displayed.

Figure 7. Representation of the narrow prep gradient chosen for the purification of the compound eluting at 4.06 min, with improved separation showing the isolated peak at 3.74 min collected.

Figure 8. Overlay of the chromatograms of the two masses that were co-eluting earlier, showing the improved separation that was achieved. Spectra highlight the success also.

step 3: fraction analysis

With the first two steps of the process complete, the user can also

decide to analyze the fractions (Figure 9). autopurify creates a

sample list containing the fractions required for analysis and auto-

matically runs them.

Time2.50 5.00 7.50 10.00

%

0

50

100

1.3e+007MS ES+ :358.1 (3)100%357.1

Time2.50 5.00 7.50 10.00

%

0

50

100

Time2.50 5.00 7.50 10.00

%

0

50

100

1.6e+007MS ES+ :TIC (3)100%357.1

Time2.50 5.00 7.50 10.00

%

0

50

100

Time2.50 5.00 7.50 10.00

%

0

50

100

1.3e+007MS ES+ :358.1 (3)100%357.1

Time2.50 5.00 7.50 10.00

%

0

50

100

Time2.50 5.00 7.50 10.00

%

0

50

100

1.6e+007MS ES+ :TIC (3)100%357.1

Time2.50 5.00 7.50 10.00

%

0

50

100

Figure 9. Fraction analysis post-collection, and post-fraction mixing by the injector/collector. TIC shows no other compounds present in the collection vessel.

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36

To ensure that the portion of the sample taken for analytical

analysis is representative of the entire collected fraction, it may

be necessary to pre-mix fractions prior to injection (done with the

injector/collector). once homogenized, analysis can be performed

on an analytical scale.

automating the process

automation of the three-step purification process is accomplished

through autopurify.

a FractionLynx browser is created after each of the three stages to

display results of the analysis and to report the recommended strat-

egy for the next stage in the process. The software can automatically

create and run the list of samples that are to continue to the next

step. The user has a choice whether to allow the three stages to run

unattended, or to manually review the results of each stage and edit

the software’s decision.

The determined strategy can be adjusted as necessary by the user

through the interactive browsers that are produced. By automating

the process, decisions can be made after regular work hours, allowing

the work to continue unattended, saving time and resources.

The root name of the data, the sample id, sample list, and the

FractionLynx browser, a123, as shown in Figure 10, are edited by the

software and carried through the purification process to make sample

and results tracking easier.

analytical interpretation

FractionLynx browsers also include chromatograms and spectral

information that are not shown in this application note. The portion

of the browser file in Figure 10 shows sample purity and the prep

strategy decision that was determined after the samples were ana-

lyzed on an analytical scale.

The preparative sample list is automatically created and run after the

analytical analysis. once the purifications are complete, the results

are processed and a new FractionLynx browser report is generated

(Figure 11).

Figure 10. Browser report created after the analytical evaluation. The resulting strategy is displayed using different colors for the injection plate. Green = mass is found, purity level between 20 and 80%, and sample requires purification; and red = mass is either not found or sample is already pure enough, and purification will not be performed.

Figure 11. Purification results, indicating where the fractions were collected, including fraction volume and spectral purity. Blue = collected fraction of the sample highlighted in the injector plate, green = passed spectral purity assessment, burgundy = review required, and red = failed purity assessment.

purification process

Upon completion of the processing of the purification results, a

sample list is generated and automatic analysis of the fractions

generated is performed (Figure 12).

Figure 12. Fraction analysis results, indicating the sample purity of the collected fractions.

Page 37: Waters Medicinal Chemistry Applications Book · 3 The Role of liquid ChRomaTogRaphy and mass speCTRomeTRy in mediCinal ChemisTRy “ Medicinal chemistry is a scientific discipline

Waters Corporation 34 Maple Street Milford, MA 01757 U.S.A. T: 1 508 478 2000 F: 1 508 872 1990 www.waters.com

Waters is a registered trademark of Waters corporation. MassLynx, autopurification, autopurify, FractionLynx, and The Science of What’s possible are trademarks of Waters corporation. all other trademarks are the property of their respective owners.

©2006-2007 Waters corporation. printed in the U.S.a.June 2007 720001466eN LB-kp

fraction analysis

The final report shows the locations of the fractions, chromatograms,

and spectra. The information in the reports can then be easily exported

in different file formats such as .xml, .csv, and .tab, to easily interface

with sample handling software packages such as liquid handlers or

weighing devices.

ConClusion

This application note shows how a library of compounds can easily

and efficiently be purified using the autopurify capabilities within

the FractionLynx application Manager. The software is capable of

automating the entire purification process, from the original analyti-

cal purity assessment, to purification, and finally to the analysis of

the fractions.

autopurify allows the process to be performed intelligently.

analytical results are used to determine if the target is present and

its purity. Based on these criteria, only samples that truly require

purification continue on through the process. Samples that do not

contain the target compound, not enough of the target, or are already

pure enough can simply be excluded from purification.

The benefits of using autopurify can be measured in time savings,

reduced solvent consumption, and overall productivity gains. This is

noticeable in several main areas:

n automated evaluation of samples before purification prevents

unnecessary purification from being performed by removing

samples that do not require purification.

n computerized evaluation of samples throughout the entire

process saves analysts from having to manually review batches

between stages of the process, and enables the subsequent

analysis to be performed immediately – without waiting for

the analyst to be present.

n computerized determination of methods required during the

process saves analysts from having to make or decide which

gradients should be used to improve separations.

n The use of narrow gradients allows for the use of shorter,

more focused gradients, saving time and solvents.

n automation from stage to stage allows for unattended

operation, combining all the savings of the process.

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oV eRV ieW

The demand for the number of samples requiring purification

continues to grow. This increase requires purifications systems

to be able to run more efficiently and with less user intervention.

However, there are a number of serious, corporate concerns with

running unattended purification. These include losing samples due

to system failure, solvent leaks, overflowing waste containers, and

solvent reservoirs running dry. another concern is the verification

that the system is actually running properly and collecting frac-

tions as expected.

This application note highlights how the Waters® autopurification™

System hardware and software can be utilized to alleviate theses

concerns. examples include software tools for monitoring solvent

usage and that can monitor the number of injections without fraction

collection. We also show how the system can be efficiently shut down

in case of error to minimize the risk of sample loss.

Finally, we demonstrate how a new splitter can increase recovery

rates and how a post-fraction collector detector can be used as a

quality control monitoring tool.

disCussion

system configuration

System configurations can vary depending on customer applications

and requirements. Waters has developed a purification system based

on input from our customers.

The requirement for chemists to be able to make analytical injections

to evaluate a sample before purification led to the development of

the Waters 2767 Sample Manager, which has two separate flow paths

– one analytical, and one for preparative. a separate and additional

flow path allows for fractions to be collected onto the instrument bed

for further analysis. This injector/collector requires a solvent deliv-

ery system that is capable of delivering reproducible and accurate

MA k ING A P U R I F IC AT IO N S YS T EM MO R E RUGG E D A N D R E L IA B L E

Ronan cleary, paul Lefebvre, and Warren potts Waters corporation, Milford, Ma, U.S.

analytical and preparative flow rates. additional pumps are regularly

added to the system for other purposes, such as post-column split-

ter make-up, at-column dilution (US patent #6,790,361), off-line

column regeneration, and pre-column modifier solvent addition.

Mass spectrometry was added to further increase the selectivity and

efficiency of the systems. These components comprise the Waters

autopurification System.

solvent monitoring

The various pumps and vessels configured in a purification system

can be defined in the monitoring software. The volume of solvent

pumped from a solvent reservoir or into a waste container is moni-

tored using the solvent monitor software.

Graphical solvent level indicators allow for easy viewing of the sys-

tem status. each solvent reservoir has information specific to that

container, maximum volume, and various warning levels.

The status of the vessels is indicated by symbols, indicating that the

system is either ok, or in Warning or an acute Warning state. The

response to the warning level is determined by the administrator.

Figure 1. The mass-directed AutoPurification System consists of the 2545 Solvent Manager, 2767 Sample Manager, System Fluidics Organizer, and PDA detector.

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40

a color-coded status page is also available, and can be accessed

remotely through the remote status monitor component of the

software.

once all the solvents are defined, monitoring occurs in the back-

ground without any user interaction. any volume of solvent pumped,

either during an acquisition or while idle, will be accounted for. even

the amount of solvent used to prime the pump is monitored.

When the software monitoring the solvent vessels identifies

a solvent level that has generated a warning condition, multiple

notifications and responses can occur, such as:

n Warning notification on the instrument page

n color-coded notification on the remote monitoring software

n email condition report sent to primary responsible party

n Terminate the analysis or batch

n Secondary emails can be sent to different individuals,

notifying them of the condition of the particular system

Figure 2. Solvent monitoring interface with both graphical and numerical reporting of system status.

Figure 3. Color-coded system status page, with icons that indicate the need to refill or empty the containers.

Figure 4. Email configuration with primary and secondary email contacts.

once the administrator has been notified, they can choose to manage

the condition by emptying or refilling the containers as necessary,

or allow the software to deal with the error condition and shut the

system down safely.

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41

Figures 5 and 6. The user can partially add or remove solvents as necessary.

Shutdown software allows the user to configure a response

produced when either the warning or acute level is reached:

n Shut down immediately

n Shut down after delay

n Shut down after sample

n Shut down after batch

n ignore the warning

The shutdown procedure configured is linked to a particular shutdown

method. This allows for an orderly shut down of the system to occur,

allowing for columns to be flushed and returned to the correct condi-

tions for storage, thus reducing the risk of damage.

Tracking failures

a critical component to ensure rugged and reliable unattended

operation is to have the system be able to stop after a defined

number of consecutive samples without fraction collection. There

are various reasons why a system may not have collected frac-

tions, and yet not be in an error state, such as a blocked splitter

or MS sample cone that prevents detection, or a blocked injection

port that keeps the sample from being loaded onto the column.

User error can also be a contributing factor. incorrect information

such as mass or wavelength can also contribute to fractions not

being collected.

Figure 7. The user can define the number of injections that can occur without fraction collection before the run is ended.

additional collectors

Frequently, analysts find that compounds other than the primary

compound of interest are of importance, so it may be necessary to

capture them in a separate collector. examples include collection

of a starting material or impurities along with the primary target.

another example is collecting all the other major peaks in addition to

the primary target. This is shown in Figure 8 with a complex natural

product separation.

Figure 8. The top chromatogram shows collection of peaks detected by ELS detection. The lower chromatogram shows the peaks detected by the MS and collected by mass trigger.

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42

Figure 9. The Waters splitter is matched to column dimensions for optimized performance.

There is no such thing as a universal detector, so it is possible that

some compounds may not be detected. a waste collector can be

added to the system, enabling all column eluent not diverted for

collection earlier to be collected separately. in Figure 8, any of the

sample not collected by either the primary or the secondary collec-

tors was captured in a separate waste collector, thus minimizing the

possibility of any sample loss.

splitter performance

on any purification system where a destructive detector is being

used, a splitter is necessary to isolate a portion of the primary

flow for analysis, allowing the rest of the sample to be directed

to the fraction collector. The flow to the collector must also go

through a delay coil to prevent this much faster flow from reaching

the collector before the triggering detector has identified the peaks

to collect.

The most important requirement of the splitter is that peak shape

and resolution achieved from the column be retained in both the

low- and high-flow solvent streams. The low-flow stream is sent to

the detectors used to trigger fraction collection. if the peaks’ shapes

differ between the triggering detector and the fraction collector, the

collection of the fraction will be less than optimal. Laminar flow can

cause the peaks on the high-flow side of the system to be larger than

the peaks on the low-flow side of the system. This can contribute to

decreased recoveries and impure fractions.

We evaluated a new Waters splitter against another commercially

available splitter to highlight the improvements that have been made

with the splitter technology.

Figure 10. The upper chromatogram shows the low-flow split to the fraction trigger detector. The middle chromatogram shows the high-flow split of the sample after using another commercially available splitter to the waste detector. The lower chromatogram shows the high-flow split of the sample using the Waters splitter to the waste detector.

Figure 11. Overlay of the trigger and collected fraction trace using a Waters splitter. The collected fraction is the purple trace, and shows little or no peak dispersion.

Figure 12. Overlay of the collections with the vertical axis linked. The green trace shows what would have been missed if a non-Waters splitter had been used.

Figure 13. AutoDelay results page with delay time and results export.

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Waters Corporation 34 Maple Street Milford, MA 01757 U.S.A. T: 1 508 478 2000 F: 1 508 872 1990 www.waters.com

Collector delay time

delay time determination can be easily accomplished with the use of

autodelay software, which will perform injections to determine the

delay time and confirm injection for the determined delay time.

Figure 14 shows the effect of delay time on the amount of missed

fraction detected in the waste detector. The larger the detected

peak corresponds to a lower recovery or increased sample loss.

When the delay time is set optimally there is only a small peak, just

above the noise. But as the delay time drifts from 1 to 3 seconds

away from the optimal, the increase signal becomes more and

more substantial. The measured recovery is greater than 99% at

the optimal delay time. With the 3 seconds too early, the recovery

is only 60%.

Figure 14. Different collection delay values have different responses in the waste detector.

ConClusion

purification systems should include functionality that allows for

unattended operation such as:

n Solvent monitoring with tiered responses such as email

notification

n Solvent monitoring with intelligent shut down

n Remote system monitoring

n Secondary fraction collection for use with other detectors

n Waste collection to enhance user confidence

Flow splitters should not increase band broadening and decrease

fraction recovery rates. The new Waters flow splitters maintain

equal peak shape for both the high and low flow for optimal fraction

recovery and purity.

The autopurification System, with technology that allows for

rugged and reliable operation, is available from Waters.

Waters is a registered trademark of Waters corporation. autopurification and The Science of What’s possible are trademarks of Waters corporation. all other trademarks are the property of their respective owners.

©2007 Waters corporation. printed in the U.S.a.June 2007 720002285eN LB-kp

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inT RoduCT ion

The identification of drug metabolites following animal or human

volunteer studies is essential to the drug discovery and development

and regulatory submissions process. Traditionally, this has been

achieved by the use of liquid or gas chromatography coupled to mass

spectrometry.1,2 More recently, the use of hyphenated techniques such

as Lc/NMR and Lc/NMR/MS have become more commonplace in the

drug metabolism laboratory, allowing a more precise identification

of the site of metabolism.3,4

While Lc/NMR and Lc/NMR/MS are extremely powerful tools, they

are typically low throughput and limited in sensitivity. The capacity

of analytical columns restricts the amount of material that can be

loaded on to the column before the column exhibits either volume

or mass overloading effects and the chromatographic resolution

is lost. Thus Lc/NMR is less attractive for the analysis of highly

potent compounds dosed at low levels or those compounds that

undergo extensive metabolism. in such cases, it is often necessary

to perform a pre-concentration step, such as Spe or liquid/liquid

extraction, both of which are time consuming and run the risk of

losing of valuable information.

The use of MS-directed purification, using semi-preparative scale

columns (typically 19 mm i.d.), is now commonplace within the

pharmaceutical industry, especially to support lead candidate

purification. This approach has also been applied to the isolation

of drug metabolites with some success.5 The extra sensitivity and

selectivity of MS/MS mass spectrometry allows for more precise

selection of drug metabolites. Furthermore, the use of neutral loss

and precursor ion scanning detection modes facilitates the collec-

tion of drug metabolites without the need for prior knowledge of

compound metabolism.

T H E A P P L IC AT IO N O F M S / M S D I R EC T E D P U R I F IC AT IO N T O T H E I D EN T I F IC AT IO N O F D RUG M E TA BO L IT E S IN B IO LOGIC A L F LU I DS

paul Lefebvre, Robert plumb, Warren potts, and Ronan cleary Waters corporation, Milford, Ma, U.S.

This application note shows how tandem quadrupole mass spec-

trometry has been employed for the isolation of the metabolites of

common pharmaceuticals from urine. The application also demon-

strates different modes of data acquisition, including scan, MRM,

constant neutral loss, and precursor ion. We also demonstrate how

the use of MS/MS-directed purification facilitates the combination of

samples from several chromatographic runs.

meT hods and disCussion

a Waters® alliance® HT System was used with a SunFire™ c18 5 µm

4.6 x 100 mm column at 40 °c. eluent flow was split 1:20 with a

Valco tee. 95% of the flow passed the 2996 photodiode array (pda)

detector to the Fraction collector iii. The other 5% of the flow was

routed directly to the Quattro micro™ Mass Spectrometer equipped

with an eSci® multi-mode ionization source.

Figure 1. The Alliance HT System with the Quattro micro Mass Spectrometer.

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46

Caffeine metabolites methods

separation

Water/acetonitrile in 0.1% formic acid, 1.25 mL/min total

flow gradient. 0 to 5 min: 0%; 5 to 35 min: 0 to 10% B; 35 to

35.5 min: 10 to 95% B; 35.5 to 39.5 min: 95% B; 39.5 to 40 min:

95 to 5% B; 45 minutes end.

ms detection

electrospray positive, 3 kV capillary voltage, 30 V cone voltage,

20 V collision energy (for MS/MS experiments).

metabolites of interest

Figure 2 shows a portion of the caffeine metabolism pathway by

demethylation.6 Target metabolites maintain the methyl group in

the 1 position. They also have a common fragment ion, m/z 57.

ibuprofen metabolites

separation

Water/acetonitrile/10 mM ammonium formate, 1.25 mL/min total

flow gradient. 0 to 5 min: 5%; 5 to 35 min: 5 to 60% B; 35 to 35.5

min: 60 to 95% B; 35.5 to 39.5 min: 95% B; 39.5 to 40 min: 95

to 5% B; 45 minutes end.

Paraxanthine, m/z 1811,7 Dimethylxanthine

Caffeine, m/z 1951,3,7 Trimethylxanthine

Theophylline, m/z 1811,3 Dimethylxanthine

1Methylxanthine, m/z 167

Common fragmentof xanthine a methylin position 1, m/z 57

Figure 2. Metabolism of caffeine by demethylation: metabolites that maintain the methyl group in the 1 position have a common fragment ion, m/z 57.

ms detection

electrospray negative, 3 kV capillary voltage, 30 V cone voltage,

20 V collision energy.

metabolites of interest

Figure 3 shows the fragmentation patterns of the ibuprofen

gluceronide metabolite.7

single quadrupole directed purification

With single quadrupole directed purification, all ions generated in

the source are passed through the quadrupole and detected. This is

possible on the Quattro micro Mass Spectrometer by using the scan

mode of acquisition. only MS1 is scanned and there is no collision

energy or scanning of Q3.

Because all of the ions generated are detected in this mode,

complex mixtures can contain numerous isobaric interferences.

consequently, multiple fractions can be generated from a single

m/z value. Figure 4 shows the collection of the caffeine metabolites

with m/z 167 and 181 detected using only the first quadrupole.

There are eight fractions collected for m/z 167 and five fractions

collected for m/z 181, with additional analysis required to deter-

mine the fraction of interest.

Figure 3. Ibuprofen gluceronide metabolite with a common product ion of m/z 193.

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47

Figure 4. Fractionation based only on scanning the first quadrupole.

Tandem quadrupole directed purification: mRm collection

With multiple reaction monitoring (MRM) data acquisition, MS1 is

pre-selected on the precursor mass and MS2 is pre-selected on a

specific product ion, as illustrated in Figure 5.

Figure 5. MS/MS MRM data acquisition.

MS1static

MS2staticRF only

MS1static

MS2static

Collision CellRF only

(all masses pass)

By selectively detecting a product ion, the signal-to-noise ratio

is optimized, thus reducing the isobaric interference and allowing

only the target to be collected. This mode of acquisition requires

previous knowledge of the exact precursor and the exact product

ions before purification.

Figure 6 shows the MRM acquisition and collection of the caffeine

metabolites. The metabolites of interest for isolation have the transi-

tions of 181 to 134, and 167 to 110.

For a peak to be present in the MRM chromatogram, both the specific

precursor and the specific product ion need to be detected. For each

target, only one fraction was collected.

Figure 7. MS/MS constant neutral loss data acquisition.

MS1scanning

MS2scanningRF only

MS1scanning

MS2scanning

Collision CellRF only

(all masses pass)

Constant neutral loss collection

a second possible mode of fraction triggering is from constant

neutral loss acquisition. Here both MS1 and MS2 are scanned in

synchronization, as illustrated in Figure 7. When MS1 transmits a

specific precursor ion, MS2 looks for a product that is the precursor

minus the neutral loss value. if the correct product is present, it

registers at the detector. The constant neutral loss spectrum shows

only the masses of all the precursors that lose the specific mass.

Time2.50 5.00 7.50 10.00 12.50 15.00 17.50 20.00 22.50 25.00 27.50 30.00 32.50 35.00 37.50 40.00 42.50 45.00

%

0

2.50 5.00 7.50 10.00 12.50 15.00 17.50 20.00 22.50 25.00 27.50 30.00 32.50 35.00 37.50 40.00 42.50 45.00

%

0

2.50 5.00 7.50 10.00 12.50 15.00 17.50 20.00 22.50 25.00 27.50 30.00 32.50 35.00 37.50 40.00 42.50 45.00

2.50 5.00 7.50 10.00 12.50 15.00 17.50 20.00 22.50 25.00 27.50 30.00 32.50 35.00 37.50 40.00 42.50 45.00

m/z 181

m/z 167

OR

Figure 6. Fractionation based on MRM acquisition.

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48

Figure 8. Fractionation based on constant neutral loss acquisition.

Time2.50 5.00 7.50 10.00 12.50 15.00 17.50 20.00 22.50 25.00 27.50 30.00 32.50 35.00 37.50 40.00 42.50 45.00

%

0

2.50 5.00 7.50 10.00 12.50 15.00 17.50 20.00 22.50 25.00 27.50 30.00 32.50 35.00 37.50 40.00 42.50 45.00

%

0

2.50 5.00 7.50 10.00 12.50 15.00 17.50 20.00 22.50 25.00 27.50 30.00 32.50 35.00 37.50 40.00 42.50 45.00

%

0

2.50 5.00 7.50 10.00 12.50 15.00 17.50 20.00 22.50 25.00 27.50 30.00 32.50 35.00 37.50 40.00 42.50 45.00

2.50 5.00 7.50 10.00 12.50 15.00 17.50 20.00 22.50 25.00 27.50 30.00 32.50 35.00 37.50 40.00 42.50 45.00

2.50 5.00 7.50 10.00 12.50 15.00 17.50 20.00 22.50 25.00 27.50 30.00 32.50 35.00 37.50 40.00 42.50 45.00

m/z100 120 140 160 180 200

%

0

100 166.9

114.4

m/z100 120 140 160 180 200

%

0

100181.0

182.0

m/z 181

m/z 167

Neutral lossof 57 TIC

Figure 8 displays the constant neutral loss of 57 acquisition and

collection of the caffeine metabolites with m/z 167 and 181. it shows

that two fractions are collected, one for each mass. These fractions

contain the target mass and have the specific neutral loss.

applications for fraction collection from constant neutral loss acquisition

mass triggered collection

With constant neutral loss acquisition, the only peaks detected are the

ones with the loss of the specific mass, in this case, 57. depending on

the specificity of the loss, numerous ions can be detected. This leads

to complex total ion chromatograms. Therefore, when triggering by

a specific mass, the collected target must contain the precursor of

interest and have a specific neutral loss.

Collection triggered on TiC

When using this mode of acquisition and collection, all the peaks

with a specific neutral loss are collected. This functionality is valu-

able when the metabolites have a specific loss related to the drug’s

structure. it could also be used for isolating a class of metabolites

with a generic loss (e.g., sulfates (–80) or glucuronides (–176)).

The precursor mass for each fraction can then be extracted and used

to aid in the identification of the metabolites.

in the constant neutral loss example shown, collection could also

have been triggered from the total ion chromatogram (Tic). all peaks

in the –57 Tic would be collected and then additional analysis or

data review would be required to find the desired fractions.

precursor ion collection

a third mode of fraction triggering is from precursor ion acquisition,

as illustrated in Figure 9. Here, MS1 is scanning and MS2 is fixed

on a specific product ion. if the specific product ion is observed, it is

registered at the detector. The spectrum only shows the masses that

have that specific product.

Figure 9. MS/MS precursor ion data acquisition.

MS1scanning

MS2staticRF only

MS1scanning

MS2staticRF only

(all masses pass)

Collision Cell

Fraction collection from a precursor ion acquisition has to be from

the Tic, since the precursor mass is unknown. This mode of fraction

collection is valuable when the metabolites are unknown, but there is

a common fragment of the core compound that can be detected.

To illustrate the common fragment ion collection capability, Figure

10 shows the glucuronic acid conjugates collected from the ibupro-

fen urine samples using the precursor ion scan mode of m/z 193.

There are three fractions that are collected, m/z 273 (not drug-

related), m/z 397 (hydroxyglucuronide conjugate), and m/z 381

(glucuronide conjugate).

Figure 10. Fractionation based on the precursor ions of the m/z 193 TIC acquisition.

Time2.50 5.00 7.50 1 0.00 12.50 15.00 17 .50 20.00 22.50 25.00 27.50 30.00 32.50 35.00 37.50 40.00 42.50

%

7

2.50 5.00 7.50 1 0.00 12.50 15.00 17 .50 20.00 22.50 25.00 27.50 30.00 32.50 35.00 37.50 40.00 42.50

%

0

2.50 5.00 7.50 1 0.00 12.50 15.00 17 .50 20.00 22.50 25.00 27.50 30.00 32.50 35.00 37.50 40.00 42.50

%

0

2.50 5.00 7.50 1 0.00 12.50 15.00 17 .50 20.00 22.50 25.00 27.50 30.00 32.50 35.00 37.50 40.00 42.50

%

0

Time2.50 5.00 7.50 1 0.00 12.50 15.00 17 .50 20.00 22.50 25.00 27.50 30.00 32.50 35.00 37.50 40.00 42.50

%

7

2.50 5.00 7.50 1 0.00 12.50 15.00 17 .50 20.00 22.50 25.00 27.50 30.00 32.50 35.00 37.50 40.00 42.50

%

0

2.50 5.00 7.50 1 0.00 12.50 15.00 17 .50 20.00 22.50 25.00 27.50 30.00 32.50 35.00 37.50 40.00 42.50

%

0

2.50 5.00 7.50 1 0.00 12.50 15.00 17 .50 20.00 22.50 25.00 27.50 30.00 32.50 35.00 37.50 40.00 42.50

%

0

m /z200 300 400 500 600 700 800

%

0

100 273.1

274.7

m/z200 300 400 500 600

%

0

100 397.0

273.1

397.8

398.7

m/z200 300 400 500 600

%

0

100 381.2

382.0

383.4

Parents of 193 TIC

m/z 381

m/z 397

m/z 273

Page 47: Waters Medicinal Chemistry Applications Book · 3 The Role of liquid ChRomaTogRaphy and mass speCTRomeTRy in mediCinal ChemisTRy “ Medicinal chemistry is a scientific discipline

Waters Corporation 34 Maple Street Milford, MA 01757 U.S.A. T: 1 508 478 2000 F: 1 508 872 1990 www.waters.com

additional collection options

The eSci multi-mode ionization source enables both eSi +/- and apci

+/- acquisition to occur within the same run. This allows for fraction

collection to be triggered from any of the acquisition channels, thus

proving useful if the metabolites require different ionization modes.

prior to this enabling technology, the only options for collection

would be to split the sample and run in different modes, or rely upon

time-based fractionation and then analyze all the fractions by both

modes to determine the targets.

The selectivity of the eSci-enabled fraction collection process can

be further enhanced by the use of mixed triggers. This approach uses

Boolean logic strings to trigger collection from multiple data traces

(e.g., collection can occur only when Mass a is present and Mass B

is not, or a peak has to be present in two different traces at the same

time for fractionation).

ConClusion

Fraction collection with a tandem quadrupole mass spectrometer is

now possible using four different modes of data acquisition: scan,

MRM, constant neutral loss, and precursor ion, which enables

improved versatility for triggering options.

n Scan mode has the potential to increase the number of isobaric

inferences detected and collected.

n MRM mode is the most selective because it only monitors a

specific precursor/product ion transition and greatly reduces

the isobaric interferences, but requires previous knowledge of

the transition.

n constant neutral loss mode can be used for collecting a class

of compounds with a target-specific loss or a generic group loss

for a broader study, or can be used as a second filter where the

target has to have a specific mass and the neutral loss.

n collection in precursor ion mode allows for all the precursors

with a specific product ion to be collected, which is valuable

when the metabolites are unknown, but there is a common

fragment of the core compound that can be detected.

Thus, these different modes of collection add value to a wide variety

of applications previously accomplished with more laborious, time

consuming, and less specific methodologies.

References

1. ismail iM and dear GJ. Xenobiotica. 1999; 29(9): 957-967.

2. dear GJ, Mallett dN, and plumb RS. LcGc europe. 2001; 14(10): 616-624.

3. dear GJ, plumb RS, Sweatman Bc, parry pS, Robert ad, Lindon Jc, Nicholson Jk, and ismail iM. Journal of chromatography B. 2000; 748: 295-309.

4. dear GJ, plumb RS, Sweatman Bc, ayrton J, Lindon Jc, and Nicholson Jk. Journal of chromatography B. 2000; 748: 281-293.

5. plumb RS, ayrton J, dear GJ, Sweatman Bc, and ismail iM. Rapid communications in Mass Spectrometry. 1999; 13(10): 845-854.

6. Bendriss e, Markoglou N, and Wainer iW. Journal of chromatography B. 2000; 746: 331-338.

7. kearney G, et al. exact Mass MS/MS of ibuprofen Metabolites using Hybrid Quadrupole-orthogonal ToF MS equipped with a LockSpray Source. Waters application Note. 2003; 720000706eN.

Waters, alliance, and eSci are registered trademarks of Waters corporation. SunFire, Quattro micro, and T he Science of What’s possible are trademarks of Waters corporation. all other trade-marks are the property of their respective owners.

©2005-2007 Waters corporation. printed in the U.S.a.June 2007 720001129eN LB-kp

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inT RoduCT ion

chemists are constantly looking for ways to improve the overall

throughput of their purification system. Time is the limiting factor

for throughput, and there are two areas where time savings can

be achieved: the amount of time required to perform a separation,

and the amount of time between injections. Making the purification

system as efficient as possible requires optimizing and minimizing

both of these times. The challenge, however, is minimize these times

without impacting the purity and recovery of the fractions.

in this application note, we examine tools available for increasing

the overall throughput of a purification system. We will use infor-

mation from the analytical separation to optimize the purification,

and will examine the steps required between injections to then

determine the most efficient way to minimize run time.

oV eRV ieW

in order to correctly compare time-saving techniques, we first

established a baseline separation to define a standard analysis and

collection time. We purified 10 drug-like compounds with a generic

10-minute preparative gradient. This baseline analysis time was then

used as the comparison time for the analysis performed when the

different time-saving chromatographic functionalities were applied.

The major areas for improving throughput are:

n decreasing the time required for the analysis

n decreasing the time between injections

one approach for decreasing the analysis time uses shallow or narrow

gradients. approaches for decreasing the time between injections

include column regeneration techniques and automatically ending

the purification run after the desired target has been collected.

E VA LUAT ING T H E T OO L S FO R IM P ROV ING P U R I F IC AT IO N T H ROUG H P U T

paul Lefebvre, Warren potts, Ronan cleary, and Robert plumb Waters corporation, Milford, Ma, U.S.

meT hods and disCussion

Components

The Waters® autopurification™ System is comprised of:

n 2545 Binary Gradient Module (BGM)

n 2767 Sample Manager

n System Fluidics organizer (SFo)

n 2996 photodiode array detector

n 3100 Mass detector

n 515 makeup pump

n passive flow splitter, 1:1000

n all components are controlled by MassLynx™

and FractionLynx™ software

The 10-sample library consisted of various drug-like compounds

at a sample concentration of about 20 mg/mL dissolved in dMSo.

Figure 1. The Waters mass-directed AutoPurification System.

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52

The chromatographic methods used water with 0.1% formic acid as

mobile phase a, and acetonitrile with 0.1% formic acid as mobile

phase B. Methanol was used as the makeup solvent for the prepara-

tive analysis.

analytical gradient

SunFire™ c18 4.6 x 50 mm, 5 µm, 1.5 mL/min total flow gradient and

a 10-minute total run time.

generic preparative

SunFire c18 19 x 50 mm, 5 µm, 25 mL/min total flow gradient.

The same gradient table, as shown in Figure 2, was used. The only

difference was the flow rate.

narrow or shallow preparative gradient

SunFire c18 19 x 50 mm, 5 µm, 25 mL/min total gradient. The start

and end percent B composition is variable and dependant on the

sample retention time during its analytical analysis.

Figure 2. Analytical gradient table.

The time window in which the analytical sample eluted defines the

conditions for the prep run. For example, if the compound eluted at

4.04 min, then the purification method would ramp up the organic

percentage so that is was 50% at 0.5 min.

Baseline throughput

The generic gradient was used to perform the purification of 10

samples and the overall run time was measured. This time is used to

compare the improvements.

Time (minutes) Composition (%B)

0.00 to 0.5 5 to %B start

0.50 to 1.67 %B start to %B end

1.67 to 2 %B end to 95

2 to 3 95

3 to 5 end

Table 1. Narrow gradient table. See Table 2 for percent B start and end.

gradient name

analytical Retention Time

%B start

%B end

a 0.00 to 1.67 5 20

B 1.67 to 2.84 20 35

c 2.84 to 4.0 35 50

d 4.00 to 5.17 50 65

e 5.17 to 6.34 65 80

F 6.34 to 7.5 80 95

Table 2. The narrow gradients used relative to the analytical retention time.

sample Retention Time (min)

Run Time (min)

Time Between

injections (min)

1 1.18 10 2

2 5.20 10 2

3 1.35 10 2

4 4.67 10 2

5 3.18 10 2

6 2.55 10 2

7 2.41 10 2

8 5.06 10 2

9 2.02 10 2

10 2.63 10 2

Total Run Time 120 minutes

Table 3. The overall throughput with the generic gradient. The total run time was 120 minutes.

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53

narrow gradients

Narrow gradients can be used to improve preparative chromatographic

resolution.1 However, if the resolution is adequate in the analytical

separation, a shorter narrow or focused gradient can be used to

increase throughput. The short method will focus its gradient on the

same organic concentration, but in a shorter time frame.

Figure 4 shows an example of one of the 10 samples being purified

by both a generic and a narrow gradient. The target was success-

fully isolated using narrow gradient d. The results show that the

resolution is maintained over the focused section of the gradient

(the blue bracket). Note that there is a loss in resolution, as expected,

in the non-focused areas of the gradient. This would have to be con-

sidered when the compound elutes at the very beginning or end of

the focused gradient.

2.00 4.00 6.00 8.00 10.00

%

0

100

2.00 4.00 6.00 8.00 10.00

%

0

100 5.06

1.99

0.49 3.20

2.45

2.201.60

4.76

4.61

4.06

5.06

6.075.58

Time1.00 2.00 3.00 4.00 5.00

1.00 2.00 3.00 4.00 5.00

2.34

0.88

0.49

0.93

2.10

1.93

1.561.19

2.34

2.60

3.01

Generic Gradient Narrow Gradient

EIC = 270 EIC = 270

TIC TIC

Figure 4. Comparison of the 10-minute generic and the 5-minute narrow purification. The blue bracket corresponds to the focused area of the gradient, where the resolution is maintained.

Rinsing and equilibration

it is important for high-quality chromatography that the column is

rinsed and re-equilibrated with the appropriate volume of solvent,

typically defined in column volumes. insufficient rinsing can cause

carryover, and equilibration time also has a significant impact on

the overall throughput, with inadequate equilibration leading to

retention time variability, poor chromatographic peak shape, or even

sample breakthrough. The quantity of rinsing solvent is dependant

upon the sample matrix, the retentiveness of the column, and the elu-

tropic strength of the rinsing solvent. Typically, two to three column

volumes is required to rinse. For equilibration, various articles report

anywhere from three to 20 column volumes can be used.2-3

For example, a 19 x 50 mm column has a volume of about 12 mL.

Two column volumes or 24 mL of 95% B were used to flush the

column, and 60 mL of 5% B were used to re-equilibrate the column.

With the gradient flow of 25 mL/min, the flush takes about

1 minute, and the equilibration takes about 2.5 minutes.

Table 4. The overall throughput increases by 1.7 fold when incorporating narrow gradients, compared to using a generic gradient.

sample generic

Retention Time (min)

narrow gradient

narrow Retention Time (min)

Run Time (min)

Time Between

injections (min)

1 1.18 a 1.38 5 2

2 5.20 e 1.65 5 2

3 1.35 a 1.74 5 2

4 4.67 d 1.94 5 2

5 3.18 c 1.75 5 2

6 2.55 B 1.90 5 2

7 2.41 B 1.95 5 2

8 5.06 d 2.34 5 2

9 2.02 B 1.30 5 2

10 2.63 B 2.08 5 2

Total Run Time70 minutes =

1.7 fold increased Throughput Figure 3. The different narrow gradients possible to focus on either improved resolution or throughput.

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54

However, the flow rate can be elevated above optimal chromato-

graphic conditions (30 mL/min for 5 µm packing), so long as the

system can withstand the overall pressure increase. We found that the

flow could be increased to 40 mL/min, only generating an additional

1300 psi of backpressure, reducing the flush time to 0.6 min and

the re-equilibration time to 1.5 min, for a 1.5-minute savings.

off-line regeneration

To increase throughput, a regeneration pump can be used to flush

and re-equilibrate the first column off-line, while the next sample

is running on a second column.

in this method, the run is terminated at 2.5 min for the narrow

gradients, or 7 min for the generic and the next injection started.

The first column is switched off-line and its flush started, while the

second column is put in-line to receive the next sample. as men-

tioned earlier, the time required for the injection to be performed

is 2 min.

The run-time savings for a generic preparative saw a reduction of

3 min per sample, for a reduction in the total run time from 120 to

90 minutes, or a 1.2-fold savings.

The run-time savings for a narrow gradient was more significant.

injection-to-injection time was reduced from 12 min with the

generic method to 4.5 min using narrow gradients and off-line

column regeneration. This reduced the total run time from 120 to

45 minutes, a 2.7-fold savings.

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0

Time

0

100

Chromatographic run time

Flush and equilibration time

Area for potential time savings

Injection time

Figure 5. Illustration of an injection cycle with chromatographic analysis time, equilibration and flush time, and injection cycle for next injections time displayed. The area where time could potentially be saved is noted.

early termination

To further reduce the time required for analysis, a software tool can be

used to automatically end the run after the target has been collected.

The throughput improvements of this feature will be illustrated for

both generic and narrow gradients.

For either gradient approach used, once the target has finished

collecting, the gradient will stop and flush with 95% B to wash the

remaining material off the column. after a defined time of rinsing,

the column will then be re-equilibrated with the initial gradient

solvent. (Note: 2 minutes of equilibration time is performed

between injections.)

Table 5. The overall throughput improvement using the run termination function can range from a two- to three-fold increase, depending on what additional tools are used. Using the regeneration pumps saves 0.6 min per injection when compared to a single column method. This corresponds to the time required to rinse the column. The re-equilibration time is incorporated into the 2 min to make an injection.

sample generic

Run Time

generic with

Regeneration

narrow Run Time

narrow with

Regeneration

1 4.03 3.43 4.23 3.63

2 8.05 7.45 4.50 3.90

3 4.20 3.50 4.59 3.99

4 7.52 6.92 4.79 3.19

5 6.03 5.43 4.60 4.00

6 5.40 4.80 4.75 4.15

7 5.26 4.66 4.80 4.20

8 7.91 7.31 5.19 4.59

9 4.97 4.27 4.15 3.55

10 5.48 4.88 4.93 4.33

Total Run Time

58.75 min =

2.0 Fold increased

Throughput

52.75 min =

2.3 Fold increased

Throughput

46.53 min =

2.6 Fold increased

Throughput

40.53 min =

3.0 Fold increased

Throughput

Page 52: Waters Medicinal Chemistry Applications Book · 3 The Role of liquid ChRomaTogRaphy and mass speCTRomeTRy in mediCinal ChemisTRy “ Medicinal chemistry is a scientific discipline

55

optimized injection routine

Throughput can be further improved by reducing the time between

injections. The injection cycle can be divided into three segments:

n aspiration of the sample into the needle

n dispensing the sample into the loop

n Washing the assembly

optimizing the speed of the aspiration enables the sample to be

quickly drawn into the needle and holding loop. care must be taken

to ensure the increased syringe speed does not create air bubbles

in the system.

once the sample has been drawn into the holding loop, it is dis-

pensed at an optimized flow rate. care must again be taken to

ensure that a high-pressure condition does not occur by operating

the syringe too quickly.

Tool original

Total Run Time

optimized injection Total

Run Time

default injection Routine

overall increase with optimized injection Routine

Generic 120 104 — 1.2

Generic + end Run

58.75 53.75 2.0 2.2

Generic + end Run + Regeneration

52.75 36.75 2.3 3.3-Fold increased Throughput

Narrow 70 54 1.7 2.2

Narrow + end Run

46.53 41.63 2.6 2.9

Narrow + end Run + Regeneration

40.53 24.53 3.0 4.9-Fold increased Throughput

Table 6. Using optimized injection routines can improve the overall throughput. The improved injection routine has a greater impact when using regeneration because the 2 min for the normal injection is used to re-equilibrate with a single column. But with regeneration, the re-equilibration is done off-line and the injection time is dead time.

Two options are available for positioning the sample in the loop. The

default setting is to center the sample in the loop, but the sample

centering can be disabled to allow the sample to be more quickly

loaded onto the front of the sample loop.

When sample centering is removed, it is possible to operate with only

one wash solvent and to be able to perform this wash at the beginning

of the injection sequence, decreasing the injection time.

Cumulative time-savings

The time required to inject and rinse was reduced from 2 min with

the standard partial loop injection to 0.4 min with the new settings.

Table 6 shows the throughput possible by combining optimized

injection settings with the various other tools.

Page 53: Waters Medicinal Chemistry Applications Book · 3 The Role of liquid ChRomaTogRaphy and mass speCTRomeTRy in mediCinal ChemisTRy “ Medicinal chemistry is a scientific discipline

Waters Corporation 34 Maple Street Milford, MA 01757 U.S.A. T: 1 508 478 2000 F: 1 508 872 1990 www.waters.com

Waters is a registered trademark of Waters corporation. FractionLynx, MassLynx, SunFire, and The Science of What’s possible are trademarks of Waters corporation. all other trademarks are the property of their respective owners.

©2007 Waters corporation. printed in the U.S.a.June 2007 720001696eN LB-kp

ConClusion

Throughput can be increased by about five-fold using a combina-

tion of narrow gradients, early run termination, off-line column

regeneration, and an optimized injection routine. This correlates to

an 80 percent decrease in run time.

n Narrow gradients can be used to improve throughput,

but require additional information about the target.

n off-line column regeneration has a greater impact on

throughput as the run time is reduced.

n early run termination improves throughput and reduces the

amount of consumed solvent saving both time and money.

n optimizing the wash sequence and adjusting when it is

performed will save additional time between injections.

n Various combinations of throughput-enhancing tools can

be used based on the specific requirements.

References

1. p Lefebvre, a Brailsford, d Brindle, c North, R cleary, W potts iii, BW Smith, Waters poster presentation, pittcon. 2003.

2. a.p. Schellinger, p.W. carr, Journal of chromatography a. 2006; 1109: 253-266.

3. Ud Neue, american Laboratory. 1997; March.

Page 54: Waters Medicinal Chemistry Applications Book · 3 The Role of liquid ChRomaTogRaphy and mass speCTRomeTRy in mediCinal ChemisTRy “ Medicinal chemistry is a scientific discipline

inT RoduCT ion

Recent advances in purification technology have shifted the through-

put bottleneck from purifying samples to fraction drying. Some of

the technologies employed for sample drying include vacuum cen-

trifugation, heated nitrogen blow-down, and lyophilization. However,

each one has the same rate limiting factor – the quantity of water

present. This quantity is dependant on the separation technique

used to generate the fractions. The most commonly used technique

is reverse phase- (Rp-) HpLc, which can generate fractions with the

water content as great as 95%.

one approach experimented with is to collect fractions directly onto

solid phase extraction (Spe) cartridges. in theory this method is

perfect, but making it automated and rugged has continued to be

a challenge. a drawback to this approach is that a very high flow

dilution pump is required to trap the compound on the cartridge. This

high flow rate requires a large quantity of sorbent with large volume

cartridges, and generates large volume fractions. another problem

with collection onto Spe cartridges is the possible change in selectiv-

ity that could result in poor trapping or breakthrough of the analyte.

This application note shows the development and optimization of a

method that removes the water and reduces the overall volume of the

collected fraction. This method works by injecting and trapping the

previously collected fraction onto a preparative column. The fraction

is trapped by diluting the loading flow with 100% aqueous mobile

phase. after the trapping has been completed, 100% organic mobile

phase is passed through the column to elute the sample. collection of

the target is triggered by the MS detector and the collected fraction

is now in 100% organic mobile.

A NOV E L A P P ROAC H FO R R E DU C ING F R AC T IO N D RY DOW N T IM E

paul Lefebvre, Ronan cleary, Warren potts, and Robert plumb Waters corporation, Milford, Ma, U.S.

meT hods and disCussion

The standard components of the Waters® autopurification™ System

were used to perform the fraction concentration. in the plumbing dia-

gram shown in Figure 1, the aqueous flow out of the gradient pump

is directed into the first tee (T1). This tee acts as a mixer, diluting the

organic concentration of the injected fraction, so that it will not break

through the trapping column. The organic flow out of the gradient

pump is directed to a second tee (T2) and is used to elute sample

from the column.

proof of principle

To establish a baseline performance of the method parameters,

10 drug-like compounds were initially purified. These purified

fractions were collected in different concentrations of organic solvent

and then used as the samples to evaluate the concentration method.

The samples were loaded onto a trapping column and eluted in 100%

organic solvent. once it was determined that the initial method was

successful, the process was optimized for minimum fraction volume

and maximum throughput. The examples shown have initial fraction

volumes as great as 30 mL of aqueous/organic and are reduced to as

little as 1.5 mL of organic solvent.

Figure 1. Plumbing diagram for the concentration system. Both fraction collection and concentration was performed on the same mass-directed AutoPurification System. Fraction collection was triggered by MS.

Page 55: Waters Medicinal Chemistry Applications Book · 3 The Role of liquid ChRomaTogRaphy and mass speCTRomeTRy in mediCinal ChemisTRy “ Medicinal chemistry is a scientific discipline

58

purification methodn 10 mg sample load

n Generic 5 to 95% gradient with water/acN/formic acid

n Fraction volume of 5 to 8 mL with recoveries of greater than 95%

Figure 4. Two of the remaining three were successful after adding base to fraction. This indicates that these samples should have been purified at a basic pH to keep the target neutral.

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

%

-2

98

m/z=235.0

1.50

0.65

%

-2

98

m/z=235.0 m/z=235.0

3.533.413.243.04 3.73 3.77 4.10

%

-2

98

m/z=235.0 m/z=235.0

4.41

4.00

0.50

4.04

4.45

4.50

0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50

0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50

%

-2

98

m/z=235.0

4.40 4.48

Time2.77

Time

Time

Time

Purification

Acid Concentration

Base Concentration(200 µL NH4OH)

Base Concentration (50 µL NH4OH)

0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50

Concentration method

The collected fractions were injected onto the same column as was

used for purification. The samples were loaded onto the column with

a loading pump at 6 mL/min 100% a, and 29 mL/min aqueous

from a dilution pump. after 6.5 min, the loading and dilution flow is

stopped. Now that the sample is retained on the column, the elution

is started at 29 mL/min of 100% B.

ResulTs

although the remaining sample was purified using the SunFire™

column, it was not retained on the column during the concentration

process. However, because fraction collection was triggered by MS,

no sample was lost. additional work is required to determine why it

was not retained.

method optimization

once the trapping method was determined to be successful, we looked

into optimizing the conditions. The parameters evaluated included

the column dimension and packing, the dilution ratio, and the elution

flow rate. an initial fraction of 10 mg of diphenhydramine collected

in 8 mL of 60% water was the concentration test sample.

Column dimensions

The column must be able to trap the target fraction and yet give a

minimum elution volume for the concentrated fraction.

The maximum flow rate and the minimum loading time were deter-

mined to establish a minimum run time. These factors are dependant

upon the column i.d., particle size, and injection loop.

19 x 50 mm trap column

5 and 10 µm packing gave the same fraction volume. The only

difference was the system back pressure.

Figure 2. Generic 5 to 95% gradient.

Figure 3. Seven of the 10 were successfully concentrated in the acidic mobile phase in which they were collected. All recoveries were greater than 85%.

Diphen

Time1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00

%

-2

98

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00

%

-2

98

m/z=167.21#1,1:1 to 1#1,1:2

012606_02a 1: Scan ES+ TIC

1.58e86.91

m/z=167.21#1,1:72

012606_01e

1: Scan ES+ TIC

1.58e8

2.74

0.710.66

2.85

Purification

Acid Concentration

Page 56: Waters Medicinal Chemistry Applications Book · 3 The Role of liquid ChRomaTogRaphy and mass speCTRomeTRy in mediCinal ChemisTRy “ Medicinal chemistry is a scientific discipline

59

Figure 6 and 7. Elution flow was reduced with minimal adjustment to peak width.

Diphen Conc 10x50 mm 5 µm column 4 mL/min load, 24 mL/min dilution

Time1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00

%

-2

98

m/z=167.21#1,1:4

013106_12b 1: Scan ES+ TIC

1.41e86.70

Elution Flow =24 mL/min

Volume = 5.8 mL

Diphen Conc 10 mm col 4 load / 22 dilution / 12 mL/min elute

Time1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00

%

-2

98

m/z=167.21#1,1:28

020106_14b 1: Scan ES+ TIC

1.31e86.79

Elution Flow =12 mL/min

Volume =2.9 mL

Figure 5. Concentration of the test fraction on a 19 x 50 mm column.

Diphen Conc

Time1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00

%

-2

98

m/z=167.21#1,1:1 to 1#1,1:2

012606_02a 1: Scan ES+ TIC

1.58e86.91

10 x 50 5 µm trap column

The overall flow rate was reduced when the method was transferred

to the 10 mm column. By reducing the elution flow rate, from

24 to 12 mL/min, the concentrated fraction volume was reduced from

8 mL to 2.9 mL.

By reducing the flow rate even further, to 8 mL/min, the original

8 mL of 60% water was reduced to 1.6 mL of 100% organic solvent.

There is minimal loss of the overall speed of the analysis with the

reduced elution flow rate. The loading and dilution pump operate

at 24 and 4 mL/min, respectively, until 6.5 min. The flow rate was

then reduced to the lower elution flow, accounting for the smaller

volume, concentrated fractions.

improving throughput

sample loading rules

n The injection volume must be less than half the volume of

the sample loop. Because the injection volume was 8 mL,

the minimum loop volume was found to be 15 mL.

n 3 to 5 times the loop volume is required to clear the sample

from the sample loop. The minimum volume found to clear

all the sample was 45 mL.

dilution ratio

The dilution ratio (dilution flow/loading flow) is a critical factor

in this method. The dilution ratio is a measure of the amount of

aqueous solvent used to dilute the fraction’s organic content to

allow it to be trapped onto the column. if the dilution ratio is too

small, it will cause breakthrough. if it is too large, it will decrease

the throughput because of the additional time required to load the

sample. Figure 9 shows the effect of the concentration with varying

dilution ratios. The results show that at a ratio of 4.5 there is a

jagged breakthrough of the target compound that is not present at

a ratio of 5 or higher.

Page 57: Waters Medicinal Chemistry Applications Book · 3 The Role of liquid ChRomaTogRaphy and mass speCTRomeTRy in mediCinal ChemisTRy “ Medicinal chemistry is a scientific discipline

60

Figure 9 A-C. Concentration of test fraction with varying dilution ratios.

Diphen Conc 10 mm col 5 load/ 25 dilution / 8 mL/min elute

Time1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00

%

-2

98

m/z=167.21#1,1:16

020206_19a 1: Scan ES+ TIC

1.12e86.96

Diphen Conc 10 mm col 4 load/ 22 dilution / 8 mL/min elute

Time1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00

%

-2

98

m/z=167.21#1,1:9

020206_14a 1: Scan ES+ TIC

1.11e86.94

Diphen Conc 10 mm col 5 load/ 20 dilution / 8 mL/min elute

Time1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00

%

-2

98

020206_10a 1: Scan ES+ TIC

1.39e86.88

m/z=167.21#1,1:40

6.61

6.47

6.256.05

7.02

Ratio = 5.5

Ratio = 5.0

Ratio = 4.5

Table 1. Relationship between loading time and total flow.

loading flow

(ml/min)

loading Time

(minutes)

dilution flow

(ml/min)

Total flow

(ml/min)

5 9.0 25 30

6 7.5 30 36

7 6.43 35 42

8 5.63 40 48

9 5.0 45 54

10 4.5 50 60

15 3.0 75 90

20 2.25 100 120

Figure 10. Results from the optimized method.

Diphen Conc 10 mm col 5 load/ 25 dilution / 8 mL/min el

Time0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50

%

-2

98

m/z=167.21#1,1:5

021406_09 1: Scan ES+ TIC

5.11e74.86

Concentration of 10 mg test fraction to under 2 mL in 5.5 min with greater than 95% recovery

scaling the method

Based on the minimum loading time and dilution ratio, it is possible

to establish the relationship between the loading time and the total

flow rate (Table 1).

To reduce the loading time to less than 5 min, the table shows that

a loading and dilution flow of 10 and 50 mL/min, respectively, are

required. This gives a total flow of 60 mL/min across the column.

handling increased back pressure

n increase the particle size: a 2x increase equals a quarter of

the back pressure

n Waters SunFire column, 10 x 50 mm, 10 µm

n 60 mL/min only generated 2,300 psi

Figure 8. Concentration of the test fraction on a 10 x 50 mm 5 µm column at an elution flow rate of 8 mL/min.

Diphen Conc 10 mm col 4 load / 22 dilution / 8 mL/min elute

Time1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00

%

-2

98

m/z=167.21#1,1:35

020106_16a 1: Scan ES+ TIC

7.57e76.89

Elution Flow =8 mL/min

Volume = 1.6 mL

Page 58: Waters Medicinal Chemistry Applications Book · 3 The Role of liquid ChRomaTogRaphy and mass speCTRomeTRy in mediCinal ChemisTRy “ Medicinal chemistry is a scientific discipline

61

example 1: 60 mg of Ketoprofen

The initial purification generated a 10 mL

fraction containing about 50% water. The

concentration method successfully reduced

the volume to 3.6 mL of organic solvent

with a recovery greater than 95%.

example 2: 20 mg phenyl-tetrazole

The purification generated two fractions

with a total volume of 18 mL contain-

ing about 60% water. The concentration

successfully reduced the volume to 3.2 mL

of organic solvent with the recovery

greater than 95%.

When the chromatography begins to

overload for the purification on a 19 x 50

mm, the fraction will not be completely

trapped on the 10 x 50 mm column.

automatic pooling

Fraction pooling on the trapping column

can also increase throughput. in Figure

13, a 3 mL fraction was collected for each

of the 10 injections. The fractions were

then individually loaded onto the trap

column and concentrated. a single 1.5 mL

fraction was collected.

Figure 11 A-B. The purification and concentration of 60 mg of ketoprofen.

Time0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00

%

0

100

Time0.250.500.751.001.251.501.752.002.252.502.753.003.253.503.754.004.254.504.755.005.255.505.75

%

0

100

Concentration:3.6 mL of organic solventwith greater than 95% recovery

Purification:10 mL fractioncontaining about 50% water

Figure 12 A-B. The purification and concentration of 20 mg of phenyl-tetrazole.

Time0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00

%

0

100

Time0.250.500.751.001.251.501.752.002.252.502.753.003.253.503.754.004.254.504.755.005.255.505.75

%

0

100

Concentration:3.6 mL of organic solventwith greater than 95% recovery

Purification:10 mL fractioncontaining about 50% water

Figure 13. An example of automatic pooling of 10 fraction tubes into a single concentrated fraction.

Time0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50

%

0

100

Time0.20 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 4.20 4.40 4.60 4.80 5.00 5.20 5.40

%

0

100

Concentration:All 10 fractions loadedon the trap columneluted togetherin 1.5 mL or organic solvent

Purification:10 injections of same sample10 3 mL fractions collected30 mL total volume

mass load

one concern with these optimized parameters is the mass load on the smaller trapping column. To evaluate this, the compounds were purified

with increasing mass load on the preparative column until overload conditions were achieved. The collected fractions were concentrated using the

optimized method. Two examples are shown.

Page 59: Waters Medicinal Chemistry Applications Book · 3 The Role of liquid ChRomaTogRaphy and mass speCTRomeTRy in mediCinal ChemisTRy “ Medicinal chemistry is a scientific discipline

Waters Corporation 34 Maple Street Milford, MA 01757 U.S.A. T: 1 508 478 2000 F: 1 508 872 1990 www.waters.com

Waters is a registered trademark of Waters corporation. autopurification, SunFire, and The Science of What’s possible are trademarks of Waters corporation. all other trademarks are the property of their respective owners.

©2007 Waters corporation. printed in the U.S.a.June 2007 720002097eN LB-kp

ConsideRaT ions

The pka of the target compound should be considered when perform-

ing purification. The target compound should be neutral during the

purification. This means that a basic compound should be run in a

basic mobile phase and, conversely, an acidic target should be run in

an acidic mobile phase.

This will result in better loading and chromatography1 and will also

ensure that the collected fraction is not ionized in solution. By being

neutral, it is more likely to be successfully trapped during the con-

centration process.

The amount of collected material, in both mass and volume, will

dictate the required system configuration. The volume of the fraction

will determine the size loop required. The mass of collected material

will determine the column size. Both the loop and column size will

determine the overall throughput of the system.

in the examples shown, all of the concentrated fractions were trig-

gered by MS. However, this was done only for method development

purposes. it is possible to collect these fractions by UV or even

just by time. When collecting by time, each tube has the same vol-

ume and organic concentration, so the time required for drying is

constant. With typical fractionation, each tube can have a different

volume and organic concentration, so the time required for drying

is variable. This variability can lead to inefficiency, by either dry-

ing for too long, or by stopping too early then checking multiple

tubes to find that you need to restart for only a few of the tubes.

BenefiTs

dry-down time

composition Volume dry down

aqueous/organic 5 to 30 mL 5 to 15 hours

100% organic 1 to 3 mL less than 30 min

concentrating the fraction to about 3 mL of organic solvent can be

accomplished in 6 min.

process enhancements

n Shorter drying times equals more efficient use of the driers.

n automatic pooling of multiple fraction tubes reduces the

post-purification sample handling.

acknowledgementsn ian Sinclair, astraZeneca

n Giovanni Gallo, Waters, Manchester, Uk

n paul Rainville, Waters, Milford, Ma

References

1. Neue Ud. Wheat Te. Mazzeo JR. Mazza cB. cavanaugh JY. Xia F. diehl dM. J. chrom. a. 2004; 1030: 123-134.

Page 60: Waters Medicinal Chemistry Applications Book · 3 The Role of liquid ChRomaTogRaphy and mass speCTRomeTRy in mediCinal ChemisTRy “ Medicinal chemistry is a scientific discipline

pRofIlIN

G

Page 61: Waters Medicinal Chemistry Applications Book · 3 The Role of liquid ChRomaTogRaphy and mass speCTRomeTRy in mediCinal ChemisTRy “ Medicinal chemistry is a scientific discipline

BaCKgRound

The physical chemistry group at a major pharmaceutical company was created to support

discovery projects in hit identification, lead identification, and lead optimization phases with

early physicochemical data. The discovery groups send test requests for selected compounds

simultaneously to respective departments via the chemical Support (cS) team within the

chemistry department. The chemistry department is where all synthesized compounds are

collected and stored. compounds are sent out for testing according to the requests, as either

standard stock solutions or solid samples.

The physical chemistry group is made up of three analytical chemists running two Lc/UV/MS

systems. These systems each consist of a Waters® alliance® HT System with a 2996 photodiode

array (pda) detector and a ZQ™ Mass detector, running on MassLynx™ Software. Testing is done in

a 96-well plate format.

among the analyses performed by the team are identification, purity, stability, and solubility tests.

id and purity evaluations are always included in all solubility and stability tests and demand addi-

tional processing of data.

T he Challenge

a screen solubility test of 48 samples took approximately

51 hours of analyst time, from when the samples were received

to when the data was entered into the database. For a plate

containing 48 duplicate samples, the variety of tasks involved:

n 4 hours doing sample prep and running the samples

n 18 hours in the office collecting compound and plate

information – codes, predicted properties, structures –

and creating appropriate sample lists

n 8 hours evaluating purity

n 19 hours doing the solubility calculations

n 2 hours inputting the final data into the company’s database.

The analyst would get results well over a week later. The physical

chemistry group recognized that tests were taking too long to deliver

results. They needed to significantly reduce bottlenecks in data man-

agement and analysis, as well as instrument resources, to improve

their ability to support discovery projects – especially as incoming

work volume was increasing.

P RO F I L E LYN X A P P L IC AT IO N MA NAG E R FO R MA S S LYN X SO F T WA R E

increasing the throughput of physicochemical profilingThe client: physical chemistry group at a major pharmaceutical company

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Waters Corporation 34 Maple Street Milford, MA 01757 U.S.A. T: 1 508 478 2000 F: 1 508 872 1990 www.waters.com

T he soluT ion

creating the proper tools for collecting sample information from

the database, formatting sample lists, and analyzing the data

generated consumed a great deal of analyst time.

By implementing profileLynx™ – a specialized application Manager

for MassLynx that automates processing of physicochemical property

analyses – into their existing Lc/MS workflow, the chemists reduced

the amount of time it takes to perform these tests from 51 to just 20

hours (Figure 1). The office time was reduced from 17 to 2.5 hours.

Because of the improved reporting capabilities of profileLynx, the

solubility evaluation now takes just 4 hours instead of 19.

Business BenefiT

While the Lc/MS sample analyses was efficient for the screen solu-

bility test, processing the data and interpreting the results required

tedious and time-consuming data manipulation and calculation. By

introducing profileLynx and other tools such as MassLynx templates

into their workflow, the customer has saved about 30 hours on the

solubility screen for each set of 48 compounds. The time is now used

in the implementation of other tests.

Waters and alliance are registered trademarks of Waters corporation. MassLynx, profileLynx, ZQ, and The Science of What’s possible are trademarks of Waters corporation. all other trademarks are the property of their respective owners.

©2006-2007 Waters corporation. printed in the U.S.a.June 2007 720001793eN LB-kp

Figure 1. Chemist’s time distribution for a screen solubility test of 48 compounds using a manual process (top) vs. ProfileLynx implementation (bottom).

0 10 20 30 40 50 60

2005ProfileLynx

2003ManualProcess

Time (hours)

Lab timeOffice timeEvaluation ID/PurityEvaluation SolubilityDatabase

as a result of the overall reduction in time, the group is able to analyze

more samples, as well as provide the critical information necessary to

make decisions about possible lead candidates more quickly.

Because of the success of profileLynx with this evaluation, the

software will be implemented with other tests within the physical

chemistry group, including solid solubility, stability, and eLogd.

WaT eRs soluT ions foR lead opT imiZaT ion

Waters System Solutions for lead optimization provide an auto-

mated, efficient selection process for determining compounds that

have potential to become successful therapeutics. These solutions

combine the strengths of Waters instruments, chemistries, software,

and customer support to assist discovery labs in characterizing

compounds faster, easier, and more cost-effectively.

Waters MassLynx software and its profileLynx application Manager

streamline data management for physicochemical property

profiling. MassLynx interfaces with upstream data systems to

build Sample Lists used for data acquisition, while profileLynx

automates the processing of chromatography-based data for

physicochemical property analysis.

Page 63: Waters Medicinal Chemistry Applications Book · 3 The Role of liquid ChRomaTogRaphy and mass speCTRomeTRy in mediCinal ChemisTRy “ Medicinal chemistry is a scientific discipline

inT RoduCT ion

The synthesis of large, focused chemical libraries allows pharmaceuti-

cal companies to rapidly screen large numbers of compounds against

disease targets. active compounds, or hits, that result from these

screens are traditionally ranked based on their activity, binding, and/

or specificity. Turning these hits into leads requires further analysis

and optimization of the compounds based upon their physicochemical

and adMe characteristics.

The critical factor to consider in physicochemical profiling is through-

put. The bottlenecks to throughput include MS method optimization

for a large variety of compounds and data management for the large

volume of data generated.

currently, experiments including solubility, chemical and biological

stability, water/octanol partitioning, paMpa, caco-2, and protein

binding are used to generate physicochemical profiles of compounds

in drug discovery. The measurement of physicochemical proper-

ties from these studies is easily enabled using chromatographic

separation and quantitation using Lc/MS/MS/UV. While the sample

analyses may be efficient, processing the data and interpreting the

results often requires tedious and time-consuming manual manipula-

tion and calculation.

This application note describes an approach to solving these prob-

lems by using MassLynx™ Software’s profileLynx™ application

Manager, a fully automated software package that allows for the

design of experiments, data acquisition, and data processing as

well as report generation.

To demonstrate the use of this software package, we have devel-

oped an automated UpLc®/MS/MS protocol for data generation.

The data acquired from multiple assays was processed by a single

processing method, all in an automated fashion. as a result, the

physicochemical profiling process was significantly simplified and

throughput increased.

A N AU T OMAT E D L C / M S / M S P ROT O CO L T O EN HA N C E T H ROUG H P U T O F P H YSICO C H EM IC A L P RO P E RT Y P RO F I L ING IN D RUG D IS COV E RY

peter alden, darcy Shave, kate Yu, Rob plumb, and Warren potts Waters corporation, Milford, Ma, U.S.

eX peRimenTal

lC conditions instrument: Waters® acQUiTY UpLc® System

column: acQUiTY UpLc BeH c18 column

2.1 x 50 mm, 1.7 µm

column temp.: 40 °c

Sample temp.: 20 °c

injection volume: 5 µL

Mobile phase a: 0.1% Formic acid in water

Mobile phase a: 0.1% Formic acid in acetonitrile

Gradient: Time a% B% curve Flow

0.00 95% 5% 6 0.60 mL/min

1.00 5% 95% 6 0.60 mL/min

1.30 0% 100% 1 0.60 mL/min

2.50 95% 5% 11 0.60 mL/min

ACQUITY TQD with the TQ Detector.

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66

ms conditions MS system: Waters TQ detector

Software: MassLynx 4.1 with profileLynx

eSi capillary voltage: 3.20 kV

polarity: positive

Source temp.: 150 °c

inter-scan delay: 20 ms

desolvation temp.: 450 °c

inter-channel delay: 5 ms

desolvation gas flow: 900 L/Hr

dwell: 200 ms

cone gas flow: 50 L/Hr

property profiling assaysn a set of 30 commercially available compounds were randomly

chosen to demonstrate the profileLynx application Manager.

n Quanoptimize™ application Manager allows for the automated

optimization of the MS multiple reaction monitoring (MRM)

conditions for each compound.

n each compound and a reference standard were analyzed by

solubility, pH stability, Logp/Logd, and microsomal stability

assays based on methods previously published.1,2,3

n For quantitative experiments, single point or multipoint

calibration curves were used.

n To mimic the current practice in discovery labs, 96-well plate

formats were used in this study.

n pH stability assays were carried out at three different pHs:

stomach (pH 1.0), blood (pH 7.4), and colon (pH 9.4).

n Solutions were shaken overnight and vacuum filtered through

a Sirocco™ plate.

n Fractions were quantified against single point 1 µM calibration

standards.

950ulBuffer/ ACN

950ul pH7.4 Buffer

950ulACN

Shake for 24 hours at 37ºC

Analyze and Quantitate

2 mM Samplesin DMSO

50 µL 50 µL 50 µL

950 µL pH7.4 buffer

950 µL buffer/ACN

950 µLACN

Shake for 24 hours at 37 °C

Centrifuge for 15 min at 3000 RPM

Dilute supernatant 1 to 100 in DMSO

Analyze and quantitate against standards

solubility

ph stability

950ul pH7.4 Buffer

950ul0.1 M HC l Ammonium

Formate

AmmoniumHydroxide

Analyze and Quantitate

Neutralize 50samples with

HC l

samples with

200 µM Samplesin DMSO

50 µL 50 µL 50 µL

950 µL 0.1 M HCl

950 µL pH 7.4 buffer

950 µL ph 9.4

ammonium formate

Sample 50 µL at times 0, 5, 19, 15, 30, and 60 min

Neutralize 50 µL samples with

450 µL, 0.02 M ammonium hydroxide

Neutralize 50 µL samples with 450 µL water

Neutralize 50 µL samples with

450 µL, 0.02 M HCl

Analyze and quantitate against standards

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67

logp/logd

50 µL sample +475 µL pH 7.4 buffer*475 µL pH 7.4 octanol**

20 µL samplesin DMSO

50 µL sample +475 µL water*475 µL octanol**

Shakeovernightat 37 °C

Manually separate organic and octanol phases

into separate vials and analyze or ...

*Octanol-saturated buffer (or water)**Water-saturated octanol

Shakeovernightat 37 °C

Inject fromoctanol phase

Inject from aqueous phase

Octanolphase

Aqueousphase

Set Alliance HTneedle depth to 18 mm to sampletop phase***

Set Alliance HTneedle depth to 0 mm to samplebottom phase***

***Using 2 mL 96-well plate

microsomal stability

T20 PlateT0 Plate

Solution A (4 °C)Phosphate buffer +

NADAPH A + NADAPH B

Solution B (37 °C)Phosphate buffer +rat liver microsomes

5 µM samplesin phosphate

buffer

Add 50 µL of5 µM sample solution +100 µL of solution A +500 µL of acetonitrile +

100 µL of solution B

50 µLof 5 µM samplesin 1 mL96-well

plate

Heat 37 °Cfor

20 min

Add 100 mLsolution A

Add 100 mLsolution B

Shake 37 °Cfor 20 min

Then add500 µL acetonitrile

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68

data processing and report generationn The profileLynx results browser contains up to three sections: a results table, the chromatogram, and the calibration curve.

n a pass/fail indicator column and user-selected highlight flags allow fast review of the data.

n The chromatogram is interactive for manual integration if needed.

solubility browser logp/logd browser

metabolic stability browser ph stability browser

Page 67: Waters Medicinal Chemistry Applications Book · 3 The Role of liquid ChRomaTogRaphy and mass speCTRomeTRy in mediCinal ChemisTRy “ Medicinal chemistry is a scientific discipline

Waters Corporation 34 Maple Street Milford, MA 01757 U.S.A. T: 1 508 478 2000 F: 1 508 872 1990 www.waters.com

Waters, alliance, acQUiTY, acQUiTY UpLc, and UpLc are regis-tered trademarks of Waters corporation. MassLynx, profileLynx, Quanoptimize, Quattro micro, Sirocco, SunFire, and The Science of What’s possible are trademarks of Waters corporation. all other trademarks are the property of their respective owners.

©2005-2007 Waters corporation. printed in the U.S.a.June 2007 720001239eN LB-kp

disCussionn The 30 compounds were analyzed with the Lc/MS/MS protocol

including MS MRM parameter optimization, MS acquisition

method creation, data acquisition, data processing, and

report generation.

n The data generated from the variety of assays were all

processed with the same software automatically.

n a single report was created for the 30 compounds that

contained results from all property profiling assays,

increasing throughput.

n Results are displayed in an interactive, graphical summary

format based on sample or experiment.

n additional improvements to throughput were achieved for

the Logp/Logd assay by utilizing the needle height adjustment

of the alliance HT system to inject directly from the two phases

of the octanol/water mixture without the need to manually

separate the two phases.

other assays supported:

n protein binding (plate or column)

n Membrane permeability (paMpa, caco-2, etc.)

n chromatographic hydrophobicity index (cHi)

n immobilized artificial membrane

ConClusion

Using the profileLynx and Quanoptimize application Managers

allows for:

n automated MS method development and data acquisition.

n a single approach for data processing and report generation

from multiple assays.

n complete and automated analysis, processing, and reporting.

n increased laboratory throughput.

References

1. kerns e. Journal of pharmaceutical Sciences. 2001; 90 (11): 1838-1858.

2. US pharmacopia. 2000; 24: 2236.

3. di L, kerns e, Hong Y, kleintop T, Mcconnell o. Journal of Biomolecular Screening. 2003; 8(X).

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optIm

IZatIo

N

Page 69: Waters Medicinal Chemistry Applications Book · 3 The Role of liquid ChRomaTogRaphy and mass speCTRomeTRy in mediCinal ChemisTRy “ Medicinal chemistry is a scientific discipline

inT RoduCT ion

once a chemical hit is found through a library screening process

and is verified, optimization of the compounds’ desired properties

takes place. This step involves an iterative process of synthesis

and reactivity measurement of the new compounds to further

develop drug candidates into the lead phase.

Because these reactions may take a long time, chemists need

to know as soon as possible if their syntheses are proceeding

as desired. This means utilizing measurement capabilities that

require minimal sample preparation and provide a fast response

giving low detection limits. another advantageous property might

be the ability to measure multiple parameters simultaneously.1

High throughput approaches can provide important time

savings in the optimization of process parameters. open access

Lc/MS is replacing TLc as a reaction monitoring tool.2 Sample

preparation of reaction mixtures can be as minimal as filtering

and dilution before injecting into the Lc/MS system. This allows

fast turnaround of results to allow the chemist to advance to the

next step.

The purpose of this application note is to demonstrate the

advantages of speed and ease of use that self-service UpLc® with

photodiode array (pda)/evaporative light scattering (eLS)/MS

detection brings to reaction monitoring studies.

S YN T H E T IC R E AC T IO N MO NIT O R ING US ING U P L C / M S

Marian Twohig, darcy Shave, paul Lefebvre, and Rob plumb Waters corporation, Milford, Ma, U.S.

Figure 1. The ACQUITY SQD for synthetic reaction monitoring.

eX peRimenTal

chromatographic separations were carried out using an acQUiTY

UpLc® System coupled to an acQUiTY® SQ Mass detector. pda and

eLS signals were collected simultaneously. Samples were analyzed

using gradients less than 1 minute. For chromatographic flexibility,

a column selection module was added.

lC conditions

Lc system: Waters® acQUiTY UpLc System

column: acQUiTY UpLc BeH c8 column

2.1 x 30 mm, 1.7 µm

column temp.: 45 °c

Flow rate: 800 µL/min

Mobile phase a: 0.1% Formic acid in water

Mobile phase B: 0.1% Formic acid in acetonitrile

Gradient: 5 to 95% B/0.7 min

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72

ms conditions

MS system: Waters SQ detector

ionization mode: eSi positive/eSi negative

capillary voltage: 3.0 kV

cone voltage: 20 V

Source temp.: 150 °c

desolvation temp.: 450 °c

desolvation gas: L/Hr

cone gas: 50 L/Hr

acquisition range: 100 to 1300 m/z

Scan speed: 10,000 amu/sec

Note: A low volume micro-tee was used to split the flow to the ELS and SQ.

els conditions

Gain: 500

N2 gas pressure: 50 psi

drift tube temp.: 50 psi

Sampling rate: 20 points/sec

pda conditions

Range: 210 to 400 nm

Sampling rate: 20 points/sec

ResulTs and disCussion

during the compound optimization stage of a discovery cycle,

medicinal chemists are not only interested in determining the key

structural features responsible for activity and selectivity, but also

what structural changes need to be made to improve these character-

istics. Because the reactions necessary to bring about these changes

may take many steps, chemists need to be sure they are progressing

as expected during the course of the reaction synthesis.

To illustrate the functionality of such a system, the synthesis of

atenolol (Figure 2) was used as a reaction model. The increase

in the formation of atenolol was monitored, as was the decrease

in the intermediate 4-hydroxyacetamide3 (Figure 3). a reaction

by-product 4-hydroxyphenylacetic acid was also observed.

Figure 2. Structures of atenolol and 4-hydroxyacetamide.

4-HydroxyphenlyacetamideC8H9NO2

Atenolol,C14H22N2O3

-- 4-Hydroxyphenlyacetic AcidC8H9O3

O

NH2

OH

O

OH

OH

Figure 3. UPLC/MS chromatograms. The reaction mixture was sampled at various time points.

4-HydroxyacetamideAtenolol

Increase Decrease

t=5 min

t=45 min

t=50 min

t=60 minTime

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73

The acQUiTY SQd is capable of scan speeds of up to 10,000

amu/sec. consequently, it is possible to employ a large number

of scan functions in a single run while still maintaining adequate

peak characterization. The fast scan speed is essential for this

functionality, as peak widths of 1 second or less are common with

the use of UpLc. Scanning multiple functions allows confirmation

of compound synthesis to be obtained on reaction components

whether they ionize in positive ion mode or negative ion mode,

eSi or apci. The total cycle time of the method was 1 minute 20

seconds, facilitating increased sample throughput.

a single run can also provide UV spectral information and an

estimation of compound purity at low wavelengths. eLS detection

is based upon the degree to which solute particles scatter light.

it has been known to give rise to similar responses for related

compounds.4 The signal can give a tentative estimation on the

relative quantities of the components present (Figure 4). it is also

an alternative detector to UV, which depends on the presence of

a chromaphore. as can be seen from Figure 4, atenolol ionizes

in eSi positive ion mode (retention time 0.28 min). The reaction

Figure 4. UPLC/PDA/ELS/MS chromatograms.

intermediate 4-hydroxyphenylacetamide ionizes in both positive

and negative ion mode (Rt 0.34 min) and 4-hydroxyphenylacetic

acid (Rt 0.39 min) only ionizes in negative ion mode.

The openLynx™ open access application Manager, part of

MassLynx™ Software, allows chemists to walk up to a terminal

and log in samples while entering the minimum information

required to run the samples.

The openLynx oaLogin screen shown in Figure 5 allows the

administrator to set up the system such that the user only needs

to input the information requested, and then upon completion,

select the Login Samples button. This will either tell the user the

designated autosampler position, or confirm the position that the

user has chosen, and ask for confirmation of position before it

will run the sample. in addition to a simplified sample submission

process, the openLynx application Manager can then process

data automatically and produce a summary report that can be

emailed or printed as desired.

The information contained in the summary report is viewed via

the openLynx browser shown in Figure 6. it clearly defines what

components are found or not found. chromatograms and spectra

are generated based on the processing parameters set up by the

administrator in the openLynx method.

Time0.10 0.20 0.30 0.40 0.50 0.60 0.70

%

0

0.10 0.20 0.30 0.40 0.50 0.60 0.70

%

0

100

100

0.10 0.20 0.30 0.40 0.50 0.60 0.70

LSU

0.000

25.000

50.000

0.10 0.20 0.30 0.40 0.50 0.60 0.70

AU

0.0

2.5e-2

5.0e-20.34

0.28

0.39

0.49

0.35

0.29

0.28

0.34

0.39

0.35

PDA

ELS

ESI Positive

ESI Negative

Figure 5. The OpenLynx single page login.

Page 72: Waters Medicinal Chemistry Applications Book · 3 The Role of liquid ChRomaTogRaphy and mass speCTRomeTRy in mediCinal ChemisTRy “ Medicinal chemistry is a scientific discipline

Waters Corporation 34 Maple Street Milford, MA 01757 U.S.A. T: 1 508 478 2000 F: 1 508 872 1990 www.waters.com

ConClusion

during the compound optimization stage of a discovery cycle,

medicinal chemists are not only interested in determining the key

structural features responsible for activity and selectivity, but also

what structural changes need to be made to improve these char-

acteristics. Because the reactions necessary to bring about these

changes may take a long time, chemists need to be sure they are

progressing as expected.

By using a walk-up UpLc/MS system, chemists were able to quickly

and easily monitor their reactions, noting the relative amounts of

starting materials and products. They were also able to note the

formation of any side products and make necessary alterations to

their reaction protocol to minimize these.

Figure 6. The OpenLynx Application Manager browser.

The described system and software combination can autonomously

evaluate large numbers of samples, with a cycle time of 1 minute 20

seconds. data can then be automatically processed and a summary

report can be generated. The scan speed capabilities of the acQUiTY

SQd make it possible to better characterize narrow chromatographic

peaks. This has become a necessity since the advent of sub-2 µm

particle technology, where chromatographic peaks can be 1 second

wide or less. The fast scan speed allows the chemist to extract as

much data as possible per injection by switching between apci and

eSi as well as positive and negative ion modes.

open access gives the chemist a walk-up system that is flexible for

analytical data acquisition. it runs as a complete system, from sample

introduction to end results.

The use of the fast-scanning MS along with the throughput of UpLc

technology allows the chemist to obtain high quality and compre-

hensive data about their compounds in the shortest possible time.

This combined with intelligent open access software allows informed

decisions to be made faster, thus supporting the needs of the modern

drug discovery process.

References

1. analysis and purification Methods in combinatorial chemistry, Wiley-interscience. (5): 87-123.

2. Lc/MS applications in drug development, Wiley-interscience. 96-106.

3. a Synthesis of atenolol using a Nitrile Hydration catalyst. organic process Research and development. 1998; 2: 274-276.

4. kibbey, c.e. Mol. diversity. 1995; i: 247-258.

Waters, acQUiTY, acQUiTY UpLc, and UpLc are registered trademarks of Waters corporation. MassLynx, openLynx, and The Science of What’s possible are trademarks of Waters corporation. all other trademarks are the property of their respective owners.

©2007 Waters corporation. printed in the U.S.a.June 2007 720002258eN LB-kp

Page 73: Waters Medicinal Chemistry Applications Book · 3 The Role of liquid ChRomaTogRaphy and mass speCTRomeTRy in mediCinal ChemisTRy “ Medicinal chemistry is a scientific discipline

BaCKgRound

a laboratory supporting the medicinal chemistry department of a large global pharmaceutical firm

relied on HpLc/MS systems in an open access environment to provide 150 synthetic chemists with

critical information about the success of their reactions. The synthetic chemists wanted to ascertain

quickly what compounds their reactions have made and whether any of the molecules are known to

be toxic.

To get the information they need, the medicinal chemists literally walk up to one of 21 open access

systems configured for the purpose, add their sample to the cue, select one of three pre-set HpLc/MS

scouting methods and walk away. Minutes later the results are emailed back to them.

at this facility, each open access system handles 600 to 700 samples per month; in 2004 the lab

ran a total of 204,000 samples, with much higher numbers expected for subsequent years.

The average run time for an HpLc/MS scouting method is 6.6 minutes. Turnaround time in this

high-throughput environment is critical. as the lab manager has said, “anything i can do to save any

amount of time, i do it.”

Challenge

The demands placed on the medicinal chemistry department for high-quality new drug candidates

dictate that speed is of utmost importance. despite this lab’s best efforts to reduce turnaround times

by pushing their HpLc methods to the limits, the wait for results was sometimes as long as an hour.

Things needed to change in order to reduce drug development timelines and cost.

despite the larger workload, the lab manager had set as his goal a five-minute – or less –

turnaround time for results. This ambitious goal clearly required a new approach.

another key concern for this laboratory manager: injection reproducibility. When chemists are track-

ing a reaction, any shift in retention times from one analysis to the other is a red flag and suggests

that something unintentional might have been created in the reactor.

T he soluT ion

in 2004, the laboratory acquired a Waters® open access acQUiTY Ultraperformance Lc® (UpLc®)

System, which they put on the front-end of a single quadrupole Waters ZQ™ Mass Spectrometer. The

goal was to see by how much they could shorten the run time of their scouting methods –without

losing sensitivity or resolution.

overall, the wait for results

has been cut in half, while

solvent consumption has

been cut by 85 percent.

T H E WAT E R S AC QU IT Y U P L C S YS T EM

Time and cost savings in an open access environmentThe client: a multi-national research-based pharmaceutical corporation

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Waters Corporation 34 Maple Street Milford, MA 01757 U.S.A. T: 1 508 478 2000 F: 1 508 872 1990 www.waters.com

By eliminating as little as one minute per analysis, the lab could save

3400 hours of total analysis time and increase the number of tests

they perform by 34,000.

Business BenefiT

The support laboratory began to see their work pay off with UpLc in

ways they hadn’t imagined. in short order, they have reduced what

was a 6.6-minute run to just 2.3 minutes, a three-fold improvement

in overall run time. Now, sub-two-second peak widths are standard

and the lab manager has reported, “i can offer my clients the same

peak capacity in one-half the time.”

overall, the wait for results has been cut in half, while solvent

consumption has been cut by 85 percent.

Moreover, the lab manager has reported getting more than 2500

injections on a single column without any degradation in results.

“i am extremely impressed with the robustness of the column –

very happy,” he has said.

perhaps the most important benefit of the open access acQUiTY

UpLc® System relates to the increase in the number of samples

expected in the near future. The lab manager anticipated an increase

of 15 to 20 percent in the next year, which would normally require

the addition of up to four complete Lc/MS systems, at a cost of over

$600,000 in capital investment. add to that increases in much-needed

laboratory space, service, and maintenance and consumables.

The lab manager has been able to develop an alternative plan to

achieve the same increase in sample capacity by replacing the inlets

on two of their existing systems with acQUiTY UpLc Systems.

This could be achieved for $120,000 in capital investment, an

80 percent savings by comparison. With no increase in lab space,

and further savings captured in consumables and solvents, the lab

now has a strengthened investment strategy for increasing capacity

and productivity going forward.

WaT eRs and uplC

The Waters acQUiTY UpLc System synergistically combines instru-

mentation, column chemistries, software for data acquisition and

processing, and support services, creating a singular solution with

superior sensitivity, resolution, efficiency, and sample throughput.

When coupled with Waters MS Technologies, UpLc provides a level of

separation, quantification, and characterization previously unattain-

able with traditional HpLc.

UpLc today is employed by companies to bring their laboratories

measurable improvements in analytical sensitivity, resolution, and

speed. Ultimately, these firms are looking for meaningful ways to

increase laboratory productivity, decrease operational costs, facili-

tate product development, and increase revenue generation.

WaT eRs open aCC ess soluT ions

Waters open access systems give chemists the ability to analyze their

own samples close to the point of production by simply walking up to

the Lc/MS system, logging their samples, placing their samples in the

system as instructed, and walking away. as soon as the analysis is

completed, sample results are emailed or printed as desired. System

configuration and setup is enabled through a system administrator who

determines login access, method selection, and report generation.

Waters, acQUiTY UpLc, acQUiTY Ultraperformance Lc, and UpLc are registered trademarks of Waters corporation. ZQ and The Science of What’s possible are trademarks of Waters corporation. all other trademarks are the property of their respective owners.

©2007 Waters corporation. printed in the U.S.a.June 2007 720001371eN LB-kp

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Waters Corporation 34 Maple Street Milford, MA 01757 U.S.A. T: 1 508 478 2000 F: 1 508 872 1990 www.waters.com

Waters, ACQUITY, ACQUITY UPLC, ACQUITY UltraPerformance LC, Alliance, ESCi, UltraPerformance LC, and UPLC are registered trademarks of Waters Corporation. ApexTrack, AutoPurification, AutoPurify, FractionLynx, i-FIT, LCT Premier, LockSpray, MassLynx, ODB, OpenLynx, ProfileLynx, Quattro micro, Quattro Premier, QuanLynx, QuanOptimize, Sirocco, SunFire, XBridge, ZQ and The Science of What’s Possible are trademarks of Waters Corporation. All other trademarks are the property of their respective owners.

©2007 Waters Corporation. Printed in the U.S.A.August 2007 720002320EN LB-KP