ITSP PAL Training - Basic

ITSP PAL Basic Course

for Lab Personnel

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Instructor

ITSP PAL Basic Course for Lab Personnelfor research purposes only

Rick YoungbloodDirector of TechnologyITSP 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

ITSP PAL Basic Course for Lab Personnel

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 ITSP2. Using the local terminal on the PAL to maintain the PAL3. Using CTC PAL Cycle Composer software (simulating supervisory control software)

to manage methods and sample lists from a PC4. A discussion of important ITSP concepts

ITSP PAL Basic Course for Lab Personnelfor research purposes only 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 PrepITSP PAL Basic Course for Lab Personnel

for research purposes only 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 needleto allow the PAL to transport the ITSP cartridge.

Septum

Media Cup

8mm Crimp SealThe crown of the ITSP cartridge seal mates with the nipple on the ITSP Lower Needle Guide to center the needle in the cartridge.


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


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.


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.


How ITSP Performs SPE

ITSP SPE MethodsITSP 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.


ITSP Hardware KitITSP 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


Solvent Reservoirs


ITSP PAL Accessories

TrayHolders



ITSP Prep Trays



ElutionTrays



Sample Trays



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


Using the PAL Local Terminal


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


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.


Sample List• Each line in the sample list represents a sample in the sequence

• Fields for controlling parameterso Method and Tray fields are selectable from a pull-down menuo Use TAB key to go to next fieldo All fields must have a valid entryo Pressing TAB – creates new lineo 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 header3. Paste (CTRL-V)


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 sampleson the tray by clicking on their positionsor clicking and dragging across multiplepositions.

• Complete selections in remaining fields.

• Finally, click the “Add” Button.


Running a Sample ListA 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”.


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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 methodis provided with the CycleComposer program. Anumber of samplingparameters can be changedto optimise the method tosuit the conditions of therun. When modified in thisway it can be stored with aunique name.


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Method Editor• Select from the list of available macros in the field labelled “Selected Macro”.

• Insert it into the field labelled “Method Macro Sequence”.


• Moving the cursor over the macro name displays a hint with a short description of the macro’s function.

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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.


• You have the option to “Run” or “Step” through a method on this screen for test purposes.

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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.

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.


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 At the beginning of each shift or assay run.

Prime the syringe – a dry syringe plunger tip may take a few samples before beginning to accurately aspirate solvents or samples

At the beginning of each shift or assay run.

Check SPE solvent levels and tray locations At the beginning of each shift or assay run.


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.


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.


Certification Test1. 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.


Additional ITSP ResourcesThe 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.


Instructor Contact InformationRick YoungbloodDirector of TechnologyITSP Solutions, Inc.706-395-8300855-395-8300 toll free

[email protected]://www.linkedin.com/in/rickyoungblood/

Mailing Address Shipping Address212 Northlake Drive 10 S. Carolina StreetHartwell, GA 30643 Hartwell, GA 30643


mailto:[email protected]

http://www.itspsolutions.com/

http://www.linkedin.com/in/rickyoungblood/

http://www.linkedin.com/in/rickyoungblood/

ITSP PAL Basic Course

for Lab Personnel

ITSP PAL Training - Basic

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