1100b B McWhinney - NSM 2014/   Brett McWhinney, Supervising Scientist, ... Workflow

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  • 18/08/2011



    AUTOMATIONBrett McWhinney, Supervising Scientist, HPLC/Mass

    Spectrometry Section, Pathology Central, Pathology Queensland

    11th August 2011AIMS NZIMLS South Pacific Congress

    Gold Coast Convention Centre


    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)



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    Misconceptions about mass spectrometryMass spectrometry is a reference methodMass 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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    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 m

    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 200L)

    Acquity BinarySolvent


    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 200L

    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



    A2 B2


    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


    Analytical Column


    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 specific