MEDICINAL CHEMIST RY AP PLICATIONS BOOK
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
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.
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.
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.
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.
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.
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.
SCREEN
ING
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.
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.
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.
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
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.
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.
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.
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.
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
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.
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.
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.
CoNfIR
matIo
N
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
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.
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.
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.
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
puRIfICa
tIoN
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.
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.
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.
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
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.
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.
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.
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.
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.
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.
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.
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.
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.
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
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.
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.
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.
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
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
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.
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.
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.
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
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.
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.
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.
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
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.
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
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.
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.
pRofIlIN
G
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
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.
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.
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
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
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
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).
optIm
IZatIo
N
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
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
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.
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
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
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
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