OUTLINE 18/08/2011 NEW DEVELOPMENTS IN LC/MS/MS FRONT-END AUTOMATION. Expanding role of tandem mass spectrometry in the clinical laboratory

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1 OUTLINE NEW DEVELOPMENTS IN LC/MS/MS FRONT-END AUTOMATION Brett McWhinney, Supervising Scientist, HPLC/Mass Spectrometry Section, Pathology Central, Pathology Queensland 11 th August 2011 AIMS NZIMLS South Pacific Congress Gold Coast Convention Centre 1. Automated method development and validation 2. Overview of sample preparation 3. Off-line automated sample preparation 4. On-line automated sample preparation 5. On-line analysis 6. Multiplexing 7. Practical examples currently in use Expanding role of tandem mass spectrometry in the clinical laboratory LC-MS/MS 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) MALDI-TOF; Q-TOF Proteomics (research, Biomarker discovery) 1

2 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 2

3 Automated method development software configuration Method development workflow Phase 1: Rapid screening using Automated Method Development Software Method Scouting. 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 ACQUITY UPLC 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 3

4 Optimization Phase 2: Method 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 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. Phase 3: Confirmation optimization results 2. Overview of sample preparation 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 All experimental workup is stored within the software for any future auditing and to meet IVD requirements 4

5 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 Principle of Solid Phase Extraction Protein Precipitation (PPT) Liquid-Liquid Extraction LLE Solid Phase Extraction SPE 5

6 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 indepth 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 3. Off-line automated sample preparation 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 racks 4 different assays (17OHP, P Mets, UFC and PF cort/pred) 3 columns 3 different temperatures 3 different mobile phase combinations and gradients Generate over 1000 results Bottle neck has moved from extraction to processing and reporting 6

7 Tecan Evo 100 Close-up view of the SPE module on a Tecan robotic system Off-line automated sample preparation Advantages 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 techniques Disadvantages 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 7

8 4. Automated on-line extraction Symbiosis system from Spark Holland Waters Acquity Online SPE Manager (OSM) Waters Acquity Online SPE System (OSM) A UPLC -enabled online solid phase extraction system Acquity Sample Manager 2 samples trays Variable injection volume (max 200µL) Acquity Column Manager 4 column capacity Modified access characteristics A UPLC -enabled online solid phase extraction system Acquity TQ Detector Acquity Binary Solvent Manager 4 Solvent Selection 15,000 psi Acquity Online SPE Manager 8

9 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 preparation Advantages 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 MS Disadvantages Limited flexibility on some systems Only perform SPE cleanup 5. On-line Analysis First Dimension - Loading Step Protein (Large molecule) Small drug molecule Salt, lipids Sample is directly injected onto the Cleanup column Analytes are retained Sample matrix flows to waste A 1 B 1 C 1 D 1 Loading Pump AS Cleanup Column waste Porous Particle 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. A 2 B 2 Eluting Pump Analytical Column Mass Spec 9

10 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 On-Line Columns A 1 B C 1 D 1 Loading Pump AS Cleanup Column waste A 2 B 2 Eluting Pump Mass Spec Analytical Column On-line analysis Advantages 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 Disadvantages Protein bound analytes must be released prior to injection onto the cleanup column Final extract is not as clean as SPE 6. Multiplexing 10

11 LC Pumps LC Pumps LC Pumps LC Pumps Increasing Throughput: Advanced Multiplexing with Multi Systems Auto sampler Four parallel LC systems, synchronized by software, all lead into the MS and work independently from each other Throughput = 60 samples/hour 2-4 LC s on a single MS/MS Column Column Column Column MS-MS Detector 0 4 min Meaningful data Peaks are sent to the detector for only 25% of the total run time leaving it idle 75% of the time. Throughput = 15 samples/hour Multiplexing Advantages 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 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 UPLC Reduces run times Accelerates method development Increases sensitivity and resolution Reduces operational costs 7. Practical examples currently in use Multiplexing 2-4 LC s on a single MS/MS Uses current MS/MS methods Up to 4x higher throughput Maintains complete flexibility Increased Productivity Increased Flexibility Increased Throughput Enhanced Data Quality Automated Sample Preparation Eliminates LLE/PPT Improves data quality Bimodal separation Minimal sample pre-extraction requirements 11

12 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 12

13 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 Automated 25(OH) vitamin D SPE method Steroids: 25-Hydroxyvitamin D Analysis A method for the analysis of 25(OH) Vit D 2 and D 3 Small sample requirement (150µL) Automated SPE using the Tecan EVO 100 Calibration range 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 13

14 Automated 25(OH) Vitamin D3 SPE method 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 Identify components Sample, labware and reagents tracking, connectivity to LIMS PPT proteins centrifugation Condition equilibrate Load supernatant wash Elute Transfer to UPLC 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 > nmol/l Inter-assay precision of 25(OH)D3 QC s (UTAK controls) at three levels (n=50) Level QC1 QC 2 QC 3 mean (nmol/l) %CV SPE recovery~70% 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. 14

15 Reading Final Thought 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 the implementation of LC-MS in the clinical laboratory. But there is no doubt that this technology is making an impact on the diagnosis and treatment of patients now and will be increasingly so in the future 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 15

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