ITSP/LC/MS/MS Opiates Method

Customized for High Throughputon a Dual Rail PAL

ITSPSolutions

AGENDA

• Introductions• ITSP Overview• Review Proposed Modifications• Open Discussion• Implement Proposed Modifications• Test-run ITSP/LC/MS/MS Opiates Method• Training• Discuss Next Steps

ITSP Team

• Kim Gamble– President of ITSP Solutions

• Thurmond Allsup– Applications Chemist at OpAns

• Rick Youngblood– Technical Director at ITSP Solutions

Project Objectives• To propose a solution for a completely automated opiates method which:• has a low capital cost,• reduces sample cycle time to increase throughput capacity without adding

workstations,• improves chromatography results through better sample preparation over

dilute and shoot– SPE concentrates analytes of interest– SPE eliminates interference compounds,

• greatly reduces maintenance on the LC/MSMS (5 to 20 times),• extends the useful life of existing capital equipment (legacy PAL and

Quantum Access),• allows reallocation of staff performing manual sample prep, and• is fully automated in a robust manner.

ITSP CartridgeAutosampler

syringe needle

Crimp seal (ITSP “crown”)

Rubber/Teflon septum

Needle GuideComponent

SPE sorbent

Media CupComponent

PAL Lower Needle Guide(modified for ITSP)

ITSP PAL Lower Needle Guide

The nipple on the bottom of the ITSP LNG mates with the crown on ITSP cartridge seal

ITSP Prep Station

96 well custom plate to hold and prep ITSP cartridges

Custom waste receptacle plumbed through MT Holder

07-UBCXP10-20A• ITSP SPE cartridge• United Chemical Technologies (UCT) Styre

Screen® BCX sorbent• Polymeric support functionalized with

Benzenesulfonic Acid(Strong Cation eXchange)

• 40-63 micron particle size• 100Å pore size• 10mg bedmass• Filter membrane above sorbent bed• 10micron PE frit below sorbent bed

Proposed Hardware Modifications• Move the Cold Stack 40mm to the right.• Remove the valve assembly from the left rail to make room for ITSP tray

holders.• Add a second Fast Wash Station on the left rail for SPE solvents.• Add two MT Holders on the left rail for ITSP prep stations.• Add ITSP drawer pulls on the left side of the Cold Stack.• Replace the left injection unit lower needle guide with the ITSP Lower

Needle Guide.• Connect a custom synchronization cable between the rails.• Connect 2 of the valves on the right rail to alternate between 2 LC’s

feeding the MS.

Dual Rail PAL Synchronization Cable

Interface 2 Interface 2DB25M DB25M

SYNC_SIG: LCINStrt Active state: High TTL-In1 1 4 TTL-Out1 Active State: High OUT_SIG: LCINStrtSYNC_SIG: LCINStop Active state: High TTL-In2 2 5 TTL-Out2 Active State: High OUT_SIG: LCINStopSYNC_SIG: StkClear Active state: High TTL-In3 3 6 TTL-Out3 Active State: Low OUT_SIG: StkBusy OUT_SIG: ITSPStrt Active state: High TTL-Out1 4 1 TTL-In1 Active State: High SYNC_SIG: ITSPStrt OUT_SIG: ITSPStop Active state: High TTL-Out2 5 2 TTL-In2 Active State: High SYNC_SIG: ITSPStop OUT_SIG: StkBusy Active state: Low TTL-Out3 6 3 TTL-In3 Active State: High SYNC_SIG: StkClear

DB25F GND 11 11 GND DB25FWash 1 Wash1 + 12 12 Pwr-Out1 + Pwr-Out1 + 12 12 Wash1 + Wash 1Wash 2 Wash2 + 14 14 Pwr-Out2 + Pwr-Out2 + 14 14 Wash2 + Wash 2

Wash1 - 24 24 Pwr-Out1 - Pwr-Out1 - 24 24 Wash1 -Wash2 - 25 25 Pwr-Out2 - Pwr-Out2 - 25 25 Wash2 -

DB25F Wash 3 Wash1 + 12Wash 4 Wash2 + 14

Wash1 - 24Wash2 - 25

Top Left Rail: ITSP Bottom Right Rail: LC-Inject

Proposed Software Modifications• Add the associated PAL objects that support the added PAL accessories.• Add the ITSP PAL objects.• Add the PAL objects that support direct synchronization.• Remove the PAL objects for the removed PAL accessories.• Modify some PAL objects needing customization for ITSP and direct

synchronization.• Add CTC PAL Loader to the supervisory computer, if needed.• Add CTC PAL Object Manager (in Service mode) to the supervisory computer, if

needed.• Add CTC PAL Cycle Composer to the supervisory computer, if needed.• Load ITSP Opiates Method macros, methods, and sample lists on the supervisory

computer.• Setup Cycle Composer to control the ITSP rail.• Setup existing PAL control software to control the LC-Inject rail (if possible).

Supervisory Control Software Options

• Native mode dual rail PAL control software available– Thermo Aria OS (not compatible with ITSP)– Axel Semrau Chronos (not as flexible nor as fast as Cycle Composer)– Gerstel Maestro (not as flexible as Chronos nor as flexible or fast as Cycle Composer)

• ITSP compatible PAL control software– CTC Analytics Cycle Composer– Axel Semrau Chronos– Gerstel Maestro– Thermo Xcalibur– Agilent MassHunter– Agilent ChemStation– AB Sciex Analyst– Any other software that can utilize ICC-CE custom cycles

We selected Cycle Composer combined with hardwired / hardcoded direct synchronization for this project because of flexibility, speed, and robust behavior.

ITSP SPE Method

• Load 200µL hydrolyzed sample• Wash with 100µL H2O• Wash with 100µL MeOH• Elute with 100µL H2O:ACN:NH2OH(7:3:0.2)• Air Flush• Inject 20µL

4 minute cycle time

Start Synchronization

ITSP Rail LC-Inj Rail

SET_OUT_SIG : ITSPStop (Off)

SET_OUT_SIG : StkBusy (On)

SET_OUT_SIG: ITSPStrt (On)

WAIT_SYNC_SIG: LCInStrt

SET_OUT_SIG : LCInStop (Off)

SET_OUT_SIG : StkBusy (Off)

SET_OUT_SIG: LCInStrt (On)

WAIT_SYNC_SIG: ITSPStrt

Cold Stack Synchronization

ITSP Rail LC-Inj Rail

WAIT_SYNC_SIG : StkClearSET_OUT_SIG : StkBusy (On)Perform ITSP SPESET_OUT_SIG: StkBusy (Off)

SET_OUT_SIG : StkBusy (Off)

WAIT_SYNC_SIG : StkClearSET_OUT_SIG: StkBusy (On)Get sample

Clean syringe

Pre clean syringe and wait at home

SET_OUT_SIG: StkBusy (Off)LC Inject and post clean syringe

Direct synchronization between the rails allows for overlapping actions, significantly increasing throughput.

Stop Synchronization

ITSP Rail LC-Inj Rail

SET_OUT_SIG : ITSPStop (On)

WAIT_SYNC_SIG: LCInStop

SET_OUT_SIG: ITSPStrt (Off)

SET_OUT_SIG : LCInStop (On)

WAIT_SYNC_SIG: ITSPStop

SET_OUT_SIG: LCInStrt (Off)

Opiates ITSP SPE Method

Opiates ITSP LC-Inj Method