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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
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