ITSP PAL Training - Basic

ITSP PAL Basic Course
for Lab Personnel

Instructor
2
ITSP PAL Basic Course for Lab Personnel
for research purposes only
Rick Youngblood
Director of Technology
ITSP Solutions, Inc.
Ga Tech Electrical Engineer and Computer Scientist
Lean Six Sigma Green Belt
30 years experience in industrial automation and controls technology including 16
years with Honeywell, Inc.
Certified by CTC Analytics in PAL Service and PAL Programming
5 years experience in ITSP PAL lab automation

This course is intended to familiarize lab personnel with the different concepts,
processes, and software programs used to run ITSP methods.
Topics include:
1. A brief overview of ITSP
2. Using the local terminal on the PAL to maintain the PAL
3. Using CTC PAL Cycle Composer software (simulating supervisory control
software) to manage methods and sample lists from a PC
4. A discussion of important ITSP concepts
3

On a PAL autosampler the ITSP
cartridge can be moved over trays or
vials to facilitate sample clean-up.
ITSP is a Consumable Solution, Not an Instrument
The heart of the ITSP system is
a patented cartridge
containing customer-defined
filter and/or SPE media.
ITSP is Instrument Top Sample Prep
4

ITSP Cartridge Components
PAL Lower Needle Guide
(modified for ITSP)
PAL Syringe Needle
SPE Sorbent or Filter Media
Small inner diameter of ITSP needle guide
- reduces volume to improve efficiency
- maintains vertical position of ITSP
cartridge during transport.
Needle Guide
Septum grips needle
to allow the PAL to transport
the ITSP cartridge.
Septum
Media Cup
8mm Crimp Seal
The crown of the ITSP cartridge seal
mates with the nipple on the ITSP
Lower Needle Guide to center the
needle in the cartridge.
5

A funnel cone beneath
the septa guides the
syringe needle into the
bore of the cartridge.
A dispersion cone
beneath the syringe
needle tip disperses
the solvent/sample
over the sorbent bed.
The needle penetration for
ITSP tray types is set by
default to 20mm.
• This allows the tip of the needle
to be positioned at the top of the
dispersion cone above the sorbent
bed.
• Longer penetration may cause the
needle to core the sorbent bed.
• Shorter penetration may not keep
the cartridge aligned properly in a
vertical orientation during
transport.
20mm
ITSP Cartridge Details
6

ITSP Cartridge Color
Product Number 10-UDAU-P is a pack of 100
(blue) ITSP SPE cartridges packed with 10mg
each of UCT Selectrasorb CSDAU sorbent.
This is a mixed-mode (SCX+RP) silica sorbent
functionalized with both benzenesulfonic
acid and C8. It is used in certain ITSP
methods to extract acidic drug analytes from
pain management patient urine samples.
Product Number 10-UDBXP-P is a pack of
100 (red) ITSP SPE cartridges packed with
10mg each of UCT Styre Screen SSDBX
sorbent. This is a mixed-mode (SCX+RP)
polymeric sorbent functionalized with both
benzenesulfonic acid and C18. It is used in
ITSP methods to extract basic and neutral
drug analytes from pain management
patient urine samples.
ITSP SPE cartridges are color coded according to the separation mode of the sorbent to
make it easier to differentiate between different phases by the lab tech. For example:
Product Number 10-BAXP-P is a pack of 100
(yellow) ITSP SPE cartridges packed with 10mg
each of Biotage Evolute AX sorbent. This is a
mixed-mode (SAX+RP) polymeric sorbent
functionalized with quaternary amine. It is used
in ITSP methods to extract acidic drug analytes
from pain management patient urine samples.
Product Number 10-UQAXU2-P is a pack of 100
(yellow) ITSP SPE cartridges packed with 10mg
each of UCT Selectrasorb CUQAX2 sorbent. This
is a mixed-mode (SAX+RP) silica sorbent
functionalized with both quaternary amine and
C8. It is used in ITSP methods to extract acidic
drug analytes from pain management patient
urine samples.
7

The analytical syringe replaces the
column sample reservoir above the
sorbent bed as found in manual SPE
cartridge formats
The needle penetrates the septum and
creates a seal so that the sample or
solvent is forced through the sorbent
bed by positive pressure when the
plunger is depressed
Solvents and sample are forced through
the SPE sorbent bed by the analytical
syringe plunger
The sample is eluted into a collection
plate and then injected into the LC
injection valve
Unlike manual SPE methods which
require a vacuum manifold, ITSP
solvent and sample flow rates can
be precisely controlled with the
syringe plunger.
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How ITSP Performs SPE

ITSP SPE Methods
ITSP SPE method templates included with ITSP startup kits demonstrate how the method
developer can allow lab personnel to easily control important cycle parameters during an assay
run. The templates include parameters to allow the tech to choose where the requisite trays
and solvent reservoirs are located on the PAL and to control the speeds at which the syringe will
aspirate and dispense solvents and samples individually.
An example of a common reverse phase ITSP SPE method might:
1. Condition the cartridge with an organic solvent to activate the
sorbent functional group(s) and clean any residual interference
compounds from the manufacturing process which might affect
chromatography.
2. Condition the cartridge with an aqueous solvent to equilibrate the
sorbent bed to the polarity of the sample matrix.
3. Load the sample onto the cartridge.
4. Wash loosely bound sample matrix interference compounds to waste
with an aqueous solvent.
5. Elute the cartridge with an organic solvent to an unused well.
6. Air flush the cartridge to evacuate dead volume in the cartridge.
7. Discard the spent cartridge.
8. Inject an aliquot of the eluate into the LC injection valve.
9. Clean the syringe.
10.Repeat the process for the next sample while the previous sample is
being analyzed.
9

ITSP Hardware Kit
ITSP Prep / Waste Station ITSP PAL Lower Needle Guide
The crown of the ITSP cartridge seal mates
with the nipple on the ITSP PAL Lower Needle
Guide to center the needle in the cartridge
10

Solvent
Reservoirs
11
ITSP PAL Accessories

Tray
Holders
12

ITSP Prep
Trays
13

Elution
Trays
14

Sample
Trays
15

ITSP Cartridge Disposal Location
Vial “ITSPdisp” - the location to dispose of spent ITSP cartridges – is most often programmed
to be located off the front end of the wash station.
16

Using the PAL Local Terminal
17
The PAL Local Terminal is used for various PAL operations, e.g., priming wash stations
or changing syringes. Please refer to the PAL User Manual for more information..

PAL Cycle Composer Software
• Sample List Editor
• Method Editor
18
PAL Cycle Composer software is used in this class to simulate similar
functionality in MassHunter to edit ITSP methods and sample lists.

Cycle Composer Levels of Control
1. Sample List provides lab personnel with access to sample lists and pre-defined
methods (similar to editing a MassHunter sample list). Sample lists run methods on
assays of case samples.
2. Method Editor provides access to pre-defined methods and to create new methods
with existing macros (similar to MassHunter PAL Method Editor). Methods run
macros with certain variables defined in the method before the run.
19

Sample List
• Each line in the sample list represents a sample in the sequence
• Fields for controlling parameters
o Method and Tray fields are selectable from a pull-down menu
o Use TAB key to go to next field
o All fields must have a valid entry
o Pressing TAB – creates new line
o Single or multiple lines can be edited
• Cut (CTRL-X)
• Copy (CTRL-C)
• Paste (CTRL-V)
• Copying a single field to a column:
1. First copy field (CTRL-C)
2. Click on column header
3. Paste (CTRL-V)
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Sample List
• By clicking on the “Trays” button an interactive screen allows the user to easily
implement a complex sample list using a graphic interface represented by a map for
each of the available trays.
• First, select the tray holder from the pull-down list. Then click on the representation
of the required tray. A map of this tray then appears showing all the available sample
positions.
• Select individual or groups of samples
on the tray by clicking on their positions
or clicking and dragging across multiple
positions.
• Complete selections in remaining fields.
• Finally, click the “Add” Button.
21

Running a Sample List
A sample list is ready to run after one or more lines are complete.
• Click the “Start” button
• The PAL will process each line until the list is completed.
• “Pause” will temporally halt the PAL without aborting the run.
• The status field will display the status of each sample, e.g. “Running”.
22

Method Editor
• Single step methods use a single macro which has the complete cycle built into it. All
values which control the cycle are shown on a single page. MassHunter can only run
single-step methods, not multi-step methods.
• A typical single step method
is provided with the Cycle
Composer program. A
number of sampling
parameters can be changed
to optimise the method to
suit the conditions of the
run. When modified in this
way it can be stored with a
unique name.
23

Method Editor
• Select from the list of available macros in the field labelled “Selected Macro”.
• Insert it into the field labelled “Method Macro Sequence”.
24
• Moving the cursor over
the macro name displays
a hint with a short
description of the macro’s
function.

Method Editor
• Adjust the macro variable parameter settings in the list on the right.
• Select “File”, then “Save Method” or “Save Method As” to save your work.
25
• You have the option to
“Run” or “Step” through a
method on this screen for
test purposes.

Method Editor
• Important! Please note that when creating an “inject-only” method in LCMS
software, and using a DLW syringe, that the standard cycle “LC-inj” is not
compatible with the DLW syringe, even if you choose a DLW syringe in the
method. Selecting “LC-Inj” as a cycle when using a DLW syringe will likely cause
the run to fail.
26
Unfortunately, MassHunter cannot
be configured to not display “LC-inj”
as a possible cycle, even if the
cycle is deleted from the hard drive,
MassHunter will reload it when
rebooted, so the operator must be
aware and remember to never
choose “LC-inj” as a cycle when
setting up a MassHunter method.
The manuals and tech support may
say to use “LC-inj”, but this is
incorrect. Instead, use a “DLW LC-
inj” cycle written specifically for use
with the DLW syringe, which can be
supplied by ITSP Solutions, Inc. if
one is not already available.

Multiple Sample Overlapping
• “Overlap” is a mechanism to reduce the overall time required to run two
or more analyses.
• One application of overlap is to schedule ITSP sample prep methods.
• The time it takes to perform an LC run of one sample can be used to
make preparations for the next sample using ITSP so that it is ready “Just-
In-Time” for the next injection.
27

Daily PAL Maintenance and Operation
Suggested daily maintenance procedures to ensure uninterrupted operation:
Maintenance Procedure Interval
Check for particles in the wash station glass
liners, bubbles in the supply lines, sufficient
flow through the valves, and sufficient wash
solvent levels
At the beginning of each shift or
assay run.
Prime the wash station
assay run.
Prime the syringe – a dry syringe plunger tip
may take a few samples before beginning to
accurately aspirate solvents or samples
assay run.
Check SPE solvent levels and tray locations
assay run.
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Important ITSP Concepts
• The front tray in a tray holder must be as tall or taller than the rear tray.
• It is very important to the successful performance of an ITSP PAL that tray objects, especially
ITSPprp#, be squarely and securely installed and then also very carefully trained after
installation. While this is an important point, minor errors in setup and position training, or
minor slippage of trays during operation, can be compensated for by the ITSP crown-nipple
mating action. The mating of the crown on the top of the cartridge seal with the nipple on the
bottom of the ITSP PAL Lower Needle Guide as the PAL approaches ITSPprp# compensates for
minor misalignment factors.
• However, the ITSP crown-nipple mating action can only compensate for one or two millimeters
of error in the X or Y axes. In addition, for ITSP transport, the PAL Lower Needle Guide is
blocked; so the PAL cannot use its Z-axis ‘seek object’ function when moving to a tray position
while transporting a cartridge.
• When training Reference Positions for the ITSP trays adjust the Z-position such that the lower
needle guide just touches the top of the ITSP cartridge, and then back off (up) one half
millimeter (0.5mm), 5 clicks.
• An ITSP cartridge should be held in position above the tray when training the Reference
Position for tray ITSPelt#.
• Septa in the seal of the ITSP cartridge are thick in order to provide a strong seal against the
needle for reliable transport and for pressurization. Therefore it is recommended to use a
needle that is 22S gauge rather than 22 gauge. The thicker barrel wall of the 22S gauge needle
is much sturdier than that of the 22 gauge needle.
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PAL Syringes for ITSP Methods
• PAL LC-Injection Valves requires a 22gauge blunt point needle (Point style “3”).
• 22S gauge syringe needles (narrow bore) help prevent coring septa, but there will
still be torn septa particles on the outside of the needle after multiple piercings.
Appropriate cleaning steps can mitigate problems from torn septa particles. Early
indicators are “sea monkeys” in solvent reservoirs and wash station glass liners. Later
indicators are plugged filters, valves, and loops.
• Hamilton X-type syringes perform better than C-line syringes.
• ILS syringes significantly perform best (available from LEAP PAL Parts).
• When installing syringes on the PAL, be careful to not side-load the plunger.
• 100µL syringes are most commonly used in ITSP methods. Larger syringes may be
used, but may not be available with 22S gauge needles.
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Certification Test
1. Place all solvent reservoirs and trays in their proper location on the PAL deck
and describe to the instructor the purpose of each location.
2. Prime the syringe.
3. Create an ITSP method to run an existing macro and test step-wise.
4. Create an ITSP sample list to run an existing method and test on 2 samples.
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Additional ITSP Resources
The USB memory stick in the ITSP startup kits
include:
• ITSP application notes
• ITSP PAL objects
• Example ITSP macros and methods
• Example ITSP automated SPE method
development macros
• ITSP setup guide
• ITSP method development guide
• Other resources such as catalogs, videos, and
presentations with more information about ITSP.
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Instructor Contact Information
Rick Youngblood
Director of Technology
ITSP Solutions, Inc.
706-395-8300
855-395-8300 toll free
Rick.Youngblood@ITSPsolutions.com
www.ITSPsolutions.com
http://www.linkedin.com/in/rickyoungblood/
Mailing Address Shipping Address
212 Northlake Drive 10 S. Carolina Street
Hartwell, GA 30643 Hartwell, GA 30643
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