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NEW DEVELOPMENTS IN LC/MS/MS FRONT-END

AUTOMATIONBrett McWhinney, Supervising Scientist, HPLC/Mass

Spectrometry Section, Pathology Central, Pathology Queensland

11th August 2011AIMS NZIMLS South Pacific Congress

Gold Coast Convention Centre

OUTLINE

1. Automated method development and validation

2. Overview of sample preparation3. Off-line automated sample preparation4. On-line automated sample preparation5. On-line analysis6. Multiplexing7. Practical examples currently in use

Expanding role of tandem mass spectrometry in the clinical laboratory

• TDM (e.g immunosuppressants, antiretroviral drugs, antidepressants)

• Drugs of abuse• Endocrinology (e.g. steroid profiles, FT3, FT4)• Screening of phaeochromocytoma (e.g. free

metanephrines)• Newborn screening (e.g. acylcarnitines, amino

acids, steroids)• Vitamin D (25-OH-D2, 25-OH-D3)• Peptidomics (Angiotensins, Oxytocin, ADH)

• Proteomics (research, Biomarker discovery)

LC-MS/MS

MALDI-TOF; Q-TOF

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Misconceptions about mass spectrometry•“Mass spectrometry is a reference method”•“Mass spectrometry is different, it will give me the right answers”

•“Mass spectrometry is accurate / precise”

•Mass spectrometry has the POTENTIAL to be all of these, but ONLY if methods are carefully developed, calibrated and validated.

•In that respect LC-MS is no different from any other analytical tool in your laboratory.

•Two main differences:–Responsibility - As long as clinical mass spectrometry remains almost entirely “home-brew” then YOU are responsible for the performance of the assays that you run.–Multiple analytes can be measured in one assay making the task even more difficult.

1. Method Development and Validation• Time consuming and difficult• Select column, solvent, buffer and pH• Change one variable at a time, laborious,

less than optimal• Systematic approach required• Method scouting where selectivity factors

such as pH, organic modifier and different column chemistry are evaluated.

• Best separation is then optimized to obtain the final desired result.

• Need to log and store all experimental data

Workflow Reverse Phase retention properties

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Automated method development software configuration

Method development workflow

Phase 1: Rapid screening using Automated Method Development Software

.

The first phase of the method development involves the screening of the major effectors of selectivity, primarily the column chemistry, buffer pH, and organic mobile phase

The method scouting protocol is outlined utilizing the ACQUITYUPLC System with the ACQUITY UPLC Column Manager and ACQUITY UPLC columns.The end goal in performing this protocol is to produce a matrix of different chromatograms that can provide information to determine the appropriate column for the application

Method Scouting

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Optimization

The software searches for the LC method that meets all the performance goals simultaneously. The best result(s) are reported along with predicted results for an experimental run

Phase 2: Method optimization

The experimental design is created using pump flow rate, gradient time, final percent organic, and column temperature as final optimization variables in the required ranges. The software creates the experimental design and exports it to Chromatography Data System, automatically creating all the necessary instrument methods, method sets, and sample sets. The experimental design is run and data processed on the chromatographic system and the results are imported back into the software.

The optimum method determined by the Software Method Optimizer was:

Column: ACQUITY UPLC BEH C8 Column, 2.1 x 100 mm, 1.7 µm

Mobile phase A: 10 mM Ammonium Acetate, pH 5.0

Mobile phase B: Methanol

Flow rate: 0.43 mL/min

Gradient: 5% to 30.0% Methanol in 9 min

Column temp.: 46 °C

Phase 3: Confirmationoptimization results

All experimental workup is stored within the software for any future auditing and to meet IVD requirements

2. Overview of sample preparation

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The Ideal Sample Preparation Method Should:

• Provide high and reproducible recoveries for acidic, basic and neutral analytes

• Be easy to use and rugged

• Be easy to automate for high sample throughput

• Removes interferences

• Be fast and cost efficient

Why do Sample Prep?• Particulates

- May limit instrument/system up-time- Plugging

• Analytes contained in a complex sample matrix- Better chromatography- Longer column lifetime- Improved accuracy and reproducibility

• Sensitivity- Analyte concentration in original sample matrix

too low to measure by instrument• Sample Matrix

- Whole blood, plasma and urine- Metabolites and structurally related compounds- Analytes bound to proteins

Protein Precipitation (PPT) Liquid-Liquid Extraction LLE Solid Phase Extraction SPE

Principle of Solid Phase Extraction

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Advantages of SPE Vs Liquid-Liquid Extraction

• Improved throughput• Decreased organic solvent usage and waste

generation• Higher and more reproducible recoveries• Cleaner extracts (contamination, solvent

impurities)• No emulsions• Tunable selectivities (SPE phase choices,

solvent mixtures)• Readily automated

NB: Protein bound analytes must be released prior to any Solid Phase extraction

Assessment of workload

• Crucial at the beginning to perform an in-depth assessment of variety and quantity of work to be performed

• The Mass Spec workload is comprised of a number of different assay (> 3) and the sample sets are small to moderate (< 100 samples)

• The Mass Spec workload is comprised of a small number of assays (≤ 3) and the sample sets are large (> 100 samples)

Automatic Column switching and Mobile Phase selection• Combination of Column manager, Sample organizer

and selectable mobile phases• Ability to run multi-assays by automatically changing

the column, mobile phase and MS conditions after each assay is finished

• Rack and stack• Typical Night

6 x 48 vial racks4 different assays (17OHP, P Mets, UFC and PF cort/pred)3 columns3 different temperatures3 different mobile phase combinations and gradients

• Generate over 1000 results• Bottle neck has moved from extraction to processing

and reporting

3. Off-line automated sample preparation

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Tecan Evo 100Close-up view of the SPE module on a Tecan robotic system

Off-line automated sample preparationAdvantages• Prepare extracts for several different

systems ie not tied to 1 LC-MS/MS• Utilised by other sections ie spread cost• Platform flexibility to perform many

different sample preparation techniquesDisadvantages• Analyst must transfer final extract to MS

and program run• Not fully automated• No communication between sample prep

and Mass Spec• Only a portion of the extract is injected

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4. Automated on-line extraction Symbiosis system from Spark Holland

Waters Acquity Online SPE Manager (OSM)

“A UPLC®-enabled online solid phase

extraction system”

Waters Acquity Online SPE System (OSM)

Acquity Sample Manager

• 2 samples trays• Variable injection volume (max 200µL)

Acquity BinarySolvent

Manager

• 4 Solvent Selection• 15,000 psi

Acquity Column Manager

• 4 column capacity

•Modified access characteristics

Acquity Online SPE Manager

Acquity TQ Detector

“A UPLC®-enabled online solid phase

extraction system”

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System Characteristics• Matrices

– Plasma, urine and other biological specimens– Injection volumes of up to 200µL

• Throughput– Governed by the longer of LC or SPE stages– LC run time is usually longest– Typically 3-5 mins with parallel processing

• Consolidation of methods on a single platform

• Multiple modes of operation– UPLC, Sample Extraction & Automated Method

Development (AMD) Mode

On-line automated sample preparationAdvantages• Fully automated• Ideal for large sample runs ie Project work• Reduced sample handling via automation• Simplified SPE protocols• Parallel processing• Greater efficiency, lower sample volumes• No evaporation or reconstitution steps• All of the analyte is eluted into the MSDisadvantages• Limited flexibility on some systems• Only perform SPE cleanup

5. On-line Analysis

Porous Particle

Salt, lipids

Protein(Large molecule)

Small drug molecule

High linear velocity, allowed by large particles, and the difference in diffusion rates cause large molecules to be excluded from interacting with column phase; passing them to waste.

First Dimension - Loading Step• Sample is directly injected onto the

Cleanup column• Analytes are retained• Sample matrix flows to waste

Loading Pump AS

Eluting Pump

MassSpec

A1B1C1D1

A2 B2

waste

Analytical Column

Cleanup Column

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Second Dimension – Analytical Separation• Eluting pump delivers strong solvent to elute the

analytes• Analytes move to analytical columns and are

separated• Detection takes place in the Mass Spectrometer

Loading Pump AS

MassSpec

Analytical Column

waste

A1 C1D1B

Eluting Pump

A2 B2

Cleanup Column

On-Line Columns

On-line analysis

• Minimizes sample preparation, inject samples directly into LC/MS system

• Reduces ion suppression, through higher specificity

• Saves time by simplifying complex sample preparation protocols

• Simplifies method development, use the same method for different matrices

• Reduces solvent consumption

Advantages

Disadvantages• Protein bound analytes must be released

prior to injection onto the cleanup column• Final extract is not as clean as SPE

6. Multiplexing

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Increasing Throughput: Advanced Multiplexing with Multi Systems

2-4 LC’s on a single MS/MS

Four parallel LC systems, synchronized by software, all

lead into the MS and work independently from each other

0

Throughput = 15 samples/hour

Meaningful data0 4 min

Peaks are sent to the detector for only 25% of the total run time leaving it idle 75% of the time.

Throughput = 60samples/hour

LC Pumps

Column

Column

Column

Column

LC Pumps

LC Pumps

LC Pumps

MS-MS Detector

Auto sampler

Multiplexing

• Accelerates results, up to 4x mass spec throughput

• Enhances productivity, analyze more samples per hour

• Improves efficiency, mass spec idle less than 4% of the time

• Increases flexibility, run up to four different assays at the same time

Advantages

Disadvantages• Increased system complexity, optimimal

performance, timing is crucial• Not suited if peaks are spread across the whole

chromatogram

Increasing LC/MS/MS Productivity with new developments in front end

•Reduces run times

•Accelerates method development

•Increases sensitivity and resolution

•Reduces operational costsUPLC

Multiplexing

Automated Sample Preparation

•2-4 LC’s on a single MS/MS •Uses current MS/MS methods•Up to 4x higher throughput•Maintains complete flexibility

•Eliminates LLE/PPT

•Improves data quality

•Bimodal separation

•Minimal sample pre-extraction requirements

•Increased Productivity

•Increased Flexibility

•Increased Throughput

•Enhanced Data Quality

7. Practical examples currently in use

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Endocrinology Applications Available

• Amines– Plasma Metanephrines

• Steroids– Vitamin D

Endocrinology Applications• Challenges

– Present at Low levels < nmol/L– Matrix interference– Lack of commercial calibration materials– Sample pre-treatment requirements– Reference methods

• Despite these challenges this is the fastest growing clinical application area today for LC/MS/MS – Selectivity and sensitivity of LC/MS/MS offers

the potential for more reliable measurement compared to other detection systems, such as immunoassays

Amines:Plasma Metanephrines

Metanephrines• Why Measure them?

– To aid the diagnosis of Phaeochromocytoma, tumour of the adrenal medulla

– Symptoms similar to hypertension– Metanephrines are catecholamine metabolites– Plasma free metanephrines have a high diagnostic

sensitivity and selectivity

• Current methodologies generally use LC / ECD– Labour-intensive, long run time– Interferences from commonly prescribed medications– Borderline sensitivity

• Required LOQ 0.10nM– Technically demanding

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Metanephrine: Off-Line Solution• Off-line SPE with LC/MS/MS• System:

– Tecan– SPE µ elution WCX and HILIC

• Enhanced selectivity and sensitivity• Run time 4 min• Excess of 200 samples/day• Elute and shoot

• Automated sample preparation– 100µL of plasma is loaded onto the LHS using barcode

tracking and automatically mixed with internal standard in a 96-well plate using the Teleshake vortex mixer module

– Samples were transferred by the LHS to a conditioned WCX Oasis µElution SPE plate and washed with water, methanol and 0.2% formic acid in acetonitrile.

Metanephrine: On-Line Solution

• On-line SPE/LC/MS/MS

• System:– Symbiosis– SPE WCX and HILIC

• Enhanced selectivity and sensitivity• Cycle time 5 min• Excess of 250 samples/day

• Minimal off-line sample preparation– 100µL of plasma – Dilution with IS 1:1

Steroids:25-Hydroxyvitamin D Analysis

Automated 25(OH) vitamin D SPE method• A method for the analysis of 25(OH) Vit D2

and D3– Small sample requirement (150µL)– Automated SPE using the Tecan EVO 100– Calibration range 5 - 250 nmol/L

• > 50 nmol/L normal

– 3.7 minute run time (injection to injection) using a BEH 2.1x50 C8 column

– Increased specificity and selectivity from MRM and UPLC

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Automated 25(OH) Vitamin D3 SPE method

Sample, labware and reagents tracking, connectivity to LIMS

Identify components

PPT proteins centrifugation

Conditionequilibrate

Load supernatant

wash

Elute Transfer to UPLC

• Sample preparation workflow using the Tecan– Approx duration 2 hr

• 96 samples prepared in 2 hrs• 288 samples prepared in a working day

– Samples prepared in 2mL 96 well plates

• All liquid handling steps executed by robot, eliminating operator error and minimising operator intervention

• Only the centrifugation step does not take place on the Tecan• Extraction plate details imported into MassLynx sample list

Linearity, Precision, Recovery

• Linearity– > 0.997 5 - 250 nmol/L

• Inter-assay precision of 25(OH)D3 QC’s (UTAK controls) at three levels (n=50)

• SPE recovery~70%

Level QC1 QC 2 QC 3mean

(nmol/L) 22 75 180%CV 6.21 4.40 3.4

Vitamin D Solution: Summary

• Low through-put manual LLE method

• High through-put semi-automated SPE method– Complete sample tracking from placing the

decapped patient tubes onto the Tecan platform

– ~300 samples per day– Significant decrease and simplification of

manual steps• Reduces operator error and intervention

Points to remember•Assess your work load and assay requirements•Front-end automation leads to bottlenecks further along the process. •Protein bound analytes must be released prior to any Solid Phase extraction•Trained staff are still required to develop and validate methods•Assay calibration & validation is just as important for mass spectrometry-based assays as any other assay that you use.•Where reference standards and reference measurement services exist use them.•Where they don’t exist, it is your responsibility to use the best available means to standardise your assay.

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Reading

• Michael Vogeser, Fabian Kirchhoff. Progress in automation of LC-MS in laboratory medicine. Clinical Biochemistry 44 (2011) 4–13

• Pierre Wallemacq. Mass spectrometry in laboratory medicine: When “high-tech” meets routine needs. Clinical Biochemistry 44 (2011) 2–3

“There are many challenges facing theimplementation of LC-MS in the clinicallaboratory. But there is no doubt that thistechnology is making an impact on thediagnosis and treatment of patients now andwill be increasingly so in the future”

Final Thought

Acknowledgements

• Steve Wilson, Solutions manager, Mass Spectrometry, Waters Australia

• Darren Jones, Product specialist LC/ LCMS Thermo Fisher Scientific

It’s great to be a Queenslander