RapidFire

high-throughput

mass spectrometry system

Users Meeting

System Tips and Tricks

Arrin Katz, Sr. Manager, Engineering

arrin.katz@agilent.com

Waldbronn, Germany

November 14, 2012

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Outline

RapidFire install base and support worldwide

Mass Spec and Rapid Fire success tips

Setup of a successful RapidFire run

Maintenance for a robust RapidFire system

Troubleshooting and hardware repairs

Advice/tips for multiple systems / users labs

Saving and re-using large batches

Standard and custom cartridges

Additional valve states

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Systems

Close to 80 units in the field

200/300/360

Geography

N. America 60%

Europe 25%

Asia 15%

Mass spectrometers

QQQ 80%

QTOF 20%

RapidFire install base

10 support engineers

ALEXIS,GERALD

BAUDAILLIER,JOE

CATBAGAN, ALAN

HARTZ, MIKE

LOZANO, STEVE

MARCHAND,SEBASTIEN

SANFORD,HOWARD

STROMAN,MAXCY

THOMSON,REBECCA

TYROS,GINA

6 support engineers

GARCIA,JUAN

LENZ,CHRISTOPH

OBERMAIER,THORSTEN

GOUJON,XAVIER

ROACH,JONATHAN

WAGNER, MORITZ

3 support engineers

QI,YI-ZHOU

WU,HAO

ZHANG,YAO-ZY

4 support engineers

KANAZAWA,YOSHINORI

KUSAKABE,TOMOKAZU

TAKAHASHI,YUTO

TAKATA,YUICHIRO

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blue: I&F trained

RapidFire/MS System

Sample

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QQQ

TOF

Agilent Confidential

December 11, 2012

RF ControlPanel

RF User Interface RF Communicator

Data

MS success tips

• Routine MS maintenance:

– Preventative maintenance

– Mass Calibration

– Cleanliness (source, Q0, Q1…)

• Proper warm up

– P3 at the assay flow rate

– QQQ: 5 minutes

– QTOF: 30 minutes

• Use high purity LC/MS grade solvents

RF success tips

• Fresh-made buffers

– Weekly (assay dependent; strongly recommend daily)

• Equilibrate the cartridges with blank injections before use

• Keep cartridge logs

– Injection counts of cartridges

– Pressure profiles

– Matrix types (neat solution, organic solution, urine, plasma….)

• Keep Instrument logs

– PM schedule (every six months)

– Repair and troubleshooting history

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Setup of a successful RapidFire run

Check vacuum presence, solvent bottles and waste flasks

Connect to the software (RF Control Panel, UI, Communicator)

Home the stages and go through the sipper configuration wizard

Check column changer is homed

Load cartridges, Go To cartridge of choice

Check valves are homed

Toggle valves (noiselessly) between load and inject

Turn pumps on and off

Verify absence of leaks or overpressures

Refer to pressure history graph

Flush Now

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Clog prevention, identification, and removal

• Sample preparation

– Spin the plate before injection (within 2 hours)

– For overnight runs, transfer supernatant after centrifuging

• Routine flushes (with aqueous solution)

• Extra flush after long sequences with organic solutions

– Move blue PEEK from pump 1 to pump 3

– Valves 1 at inject position and valve 4 at load position

– Turn on pump 3 at 8 mL/min

• Flush with both aqueous and organic solutions if a minor clog occurs

• Aspiration verification (25-40 sec/mL)

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Troubleshooting: Aspiration issues

• vacuum connections

- 70 kPa

• Sip Height deep enough?

• aspiration valves in inject,

through sipper 1 mL water in 25 - 40 s

through loop significantly faster?

• flush in both directions

with both solvents

• sip sensor calibration (“ – “ button)

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• Sapphire window (transparent, chemically inert)

• Fibers: one emits, one receives light

• bright / liquid ~ 2 X dark / air readout

• Calibration using the static “–“ button

- tube filled with water

- press “ - ” for 3 seconds to enter static teach mode

- when “ 1st ” flashes, present air, then press “ - ” briefly

- when “ 2nd ” flashes, present water, then press “ - ”

- “ pass ” will flash with contrast grade displayed (e.g. 2000)

- check with alternating full / empty wells

Troubleshooting: Sip sensor calibration

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Troubleshooting: Home a valve

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Troubleshooting: Home a valve

• Under System Tools\ Valve Tuner, press Find

with the Limited Homing box checked (60o only)

Tip: first press the E-STOP and manually

rotate the coupling counterclockwise

• Make sure load > 50 (positive)

inject < - 50 (negative)

Home 0 must be between inject and load

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Troubleshooting: Overpressure

• Determine pump that overpressured

• Check pressure as you toggle valves from

load to inject

• Replace cartridge

• Trace fluidic lines and monitor pressure as

you connect/disconnect

• Replace tubing

• Flush groove in valve

• Replace valve

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Troubleshooting:

Change a valve • disconnect tubing

loosen coupling’s bottom screw

remove (9/64 hex-key) top two screws

• V1 and V3 outward-facing sticker, V2 inward

• slide in new valve (with adapters, if easier)

tighten two screws

reconnect tubing

• Home valve

tighten coupling’s bottom screw

define valve’s load and inject positions

by rotating coupling manually

• save new configuration values

by clicking Set (underneath Cur.)

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Troubleshooting: Initialize NI boards

• Go to Start > All Programs > National Instruments > Measurement & Automation

• The Devices and Interfaces tree expands to - PCI-x (1) “linear motions”

- PCI-x (2) “rotary motions”

• Highlight each board - Reset Device

- Initialize

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• Keep a supply of spare parts in house:

• Nano-valve *** new part # G9203-80026

• RF specific fittings

• RF specific tubing

• Needles

• Cartridges

• Consult us for recommended parts and

quantities

Spare parts – minimize downtime

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RapidFire experiment building blocks

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Batch related definitions

Batch file: a .RFBAT recipe enumerating how to run one or multiple plates of a RapidFire‐

MassHunter experiment by specifying for each plate which (a) platemap to follow, and for each

sequence within each plate which (b) .RFCFG RapidFire Method, (c) solid‐phase extraction (SPE)

cartridge and (d) .M MS Method to use.

MS Method: a .M data acquisition method in MassHunter, saved in D:\ MassHunter\ Methods\

RapidFire\.

Platemap file: a .RFMAP profile of the series of sequences, and of wells within each sequence, to be

visited during the analytical read of a 96‐ or 384‐well microtiter plate during a RapidFire‐MassHunter

experiment.

RapidFire Method: a .RFCFG summary of RapidFire parameters to apply to a given sequence,

including (a) RapidFire cycle timing, (b) pump flow rates, (c) sip height, (d) occurrence of “wash

between sips” (dips of the sipper tube and its guiding needle in both wash chimneys in‐between

injections), and (e) existence of blank injections (full cycles in the organic wash chimney in‐between

sample injections).

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Multiple plates / users / mass spectrometers

Tips for lab sanity

Plate types:

• Single cfgs directory C:\ Program Files\ Agilent\ RapidFire\ cfgs

• Multiple .PLATECFG configuration files to save all parameters for all plates

- teach Twister vertical adjustment for stage and hotels

- Sipper Configuration Wizard for each plate type

Users

• Users can save batch files in separate, project-specific directories

Mass spectrometers

• Same IP address for all MS = 192.168.254.1

RF = 192.168.254.2

• RF Communicator now stops 2 .EXE processes first

RF Stop Processes new icon too

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Prepare and re-use large batches

• In Sequences mode, with barcodes as Plate Names, an exhaustive set of recipes

(= platemaps + .RFCFG methods + SPE cartridges + .M or .DAM methods)

can be saved.

• As the batch is run, only the plates with “today’s barcodes” will be read.

Other lines in the batch will be ignored, they won’t hurt or delay the run!

Methods

now linked

to MS

method dir.

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Selection chart for RapidFire SPE cartridges

Type Packing Typical Applications Part #

A C4 Small molecules, peptides, oligos G9203A

A2 C4 Small molecules, peptides, oligos G9525A

B Cyano Hydrophobic compounds G9204A

C C18 Proteins G9205A

D Hypercarb Hydrophilic compounds, small molecules G9206A

E C8 Proteins G9207A

F Phenyl Aromatic compounds G9208A

H HILIC Hydrophilic compounds, small molecules G9209A

O Blank Testing G9211A

Z Custom Custom applications G9210A

Inlet

Frit

Frit

Outlet

SPE

Packing

• Custom packed per customer specifications

• Order submitted to Agilent (minimum quantity 5, in 5 unit increments)

• Packing material shipped to Agilent Wakefield

• 45 days lead time

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Custom Cartridges – G9210A

Matrix bottles – RF300/360

Four matrix bottles: 10 mL/vial

• External standards

• QC samples

• Special wash solutions

• Calibration curve

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P/N 5190-5799 P/N 5188-9057

Plate Seals

• Agilent PlateLoc Thermal Microplate Sealer P/N G5402A

• Agilent peelable aluminum seal P/N 24210-001

• Agilent pierceable aluminum seal P/N 06644-001

• Find local support contact info at

http://www.chem.agilent.com/en-US/Contact-US/Pages/ContactUs.aspx

• Open service request (under warranty or service agreement)

– serial number, e.g. RF-MS XX (up to 57), USXXXXXXXX, or

SGXXXXXXXX

(on the instrument frame)

• Request quote for onsite services (no warranty or service contract)

• Request quote for parts and consumables

• Keep record of the service request number (SR#)

• Save log files and screenshot s of error messages

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Service contact information

User feedback and wish list

• Reduce min. volume required

• Increase unattended operation time – over weekend

• Sample prep options

• Reduce volume for matrix stations

• Temperature control

• Interface with existing lab robotics

• Provide test data for cartridges (performance variation)

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Questions and discussion

What improvements would you like to see?

What tips do you have for successful operation?

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Agilent RapidFire High-

throughput Mass

Spectrometry Software

Summary of Features

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Powerful Tools - Simple Workflow

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Minimal

Operator

Intervention

Flexible

Sample

Information

Real-time

run

monitoring

Powerful

Quantitative

Analysis

Customizable

Report

Generation

Flexible Sample Information Format

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Import sample

info from LIMS

Multiple user defined

columns available

Add method to input

file for minimal

operator intervention

Flexible Sample Information Format

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Converter allows

you to use your

own column

headers in sample

input

Set up conversion to MassHunter field headers once and

then all experiments will be processed automatically

Run Template

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Run Template = a one plate batch defining sequences, RF method, cartridge and MS method

Run Template

Minimal Operator Intervention

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1. Create run templates in advance

Minimal Operator Intervention

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2. Load Sample File

Minimal Operator Intervention

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3. Press Start

Real-time Run Monitoring

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RapidFire Injections

Real-time Run Monitoring

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Monitor the TIC in real-time with MassHunter Acquisition software

MassHunter Qual

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MassHunter Quant

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Load .d files containing all sample info,

run parameters and mass spec data

Powerful Quantitative Analysis

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Complete MassHunter Quantitative Workstation features

available on request

Customizable Report Generation

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Excel PDF