Sample prep in bio-analysis: the “last frontier” and starting point for an accurate LC-MS/MS analysis. FABIAN symposium 2006 Bio-analytical process Process: Method Development Sample Preparation Running of Samples Data Analysis Report Samples Short (no) method Process steps: Method Development Validation Assay Performance Workflow development ! Clean extracts ! ! ! Reduce Ion-suppression Sample prep techniques: • Protein Precipitation • Liquid-liquid Extraction (LLE) • Off-line SPE • Column Switching • On-line SPE Reduce carry-over LC-MS/MS Results 2 Sample Preparation Techniques Protein Precipitation Positive - Simple, generic - No Method Development time - Fast sample prep (96-well titer plate automation) Negative - Not selective, limited clean-up with a high potential of ion-suppression in LC-MS analysis - Drugs can precipitate with proteins - Centrifugation - filtration or additional clean-up steps are needed - Sample is diluted, no pre-concentration steps / low sensitivity assays 3 Sample Preparation Techniques Liquid-Liquid Extraction (LLE) Positive - Known and standard technique - Relatively simple to do as a routine - Trend to micro LLE - Low LOD’s are possible Negative - Moderate selective (limited in solvent selections) - Examine and use pH/ionic strength/temperature to get a selective extraction process - Multiple extractions needed to get the recovery - Often evaporation steps needed - Emulsions formed cause recovery loss - Difficult to automate, semi-automated steps - As routine method labour intensive 4 Sample Preparation Techniques ! ! ! ! ! ! Ev ap or at io Re n co ns tit ut io In n je ct io n ng ry i D ! El ut io n pl e as he s sa m ad ! W ! Lo ! Eq ui li So lv at io n br at io n Off-line Solid Phase Extraction (SPE) N2 ! Analytical Column Positive - Selective sample clean-up - Various SPE formats - phases - Sample preconcentration - Automated systems - Trend to 96-well micro SPE (avoids drying steps) Negative - Labour intensive method development - Evaporation steps often needed - Multiple steps, difficult sample traceability - Risk of running dry - Limited sample processing pressures - Sample prep at day-time, not a fully automated process 5 Sample Preparation Techniques Column switching, fixed pre-column Positive - (Known) LC technique Protein precipitation - (semi)-automated systems - Sample pre-concentration -Sample Fast, however pre-column Centrifugation regeneration is needed Filtration Negative - Pre-column lifetime is limited and strongly depends on regeneration / sample injection volumes - Peak focusing is needed to get small elution peaks LC-MS/MS Auto - Carry-over from stationary pre-column sampler - Sample pre-treatment is always needed (protein precipitation, filtration, centrifugation) 6 Sample preparation techniques tio n lo ad as in he g s ! ! In je c Sa m ! W Eq ui li pl e So lv at io n br at io n On-line Solid Phase Extraction (SPE) ! Switch valve Analytical Column Positive: - Disposable SPE cartridges - Minimal pre-treatment steps - Sample preparation at high pressure and controlled flow-rates - All sample is analyzed, sensitive assays - Systematic, automated method development - Fully automated and integrated process Negative: - Integrated sample prep system needs higher qualified and well trained operators - Biological samples in the LC-MS lab. - Linked to one MS system, not a workstation approach. 7 Bio-analytical process Method Development Sample Preparation Running of Samples Data Analysis Report N MS tuning LC-MD Prep-MD Validation Valid? Y Criteria: Simplicity of the developed method Short Method Development time Validation failures kept at the minimum Develop the most selective sample prep method (remove matrix components - extract analytes ) 8 Validation failures N Choice of technique Develop method Validation Valid? Y • Fast MD vs robust analytical assay " choice • Fast MD vs validation failures " conflict • Validation failures translate in time pressure - New set of conditions?? - New method?? - New sample prep technique? 9 Protein Precipitation • Only used in bio-analysis for removal of proteins • Proteins are denaturated with acid, base, salts or organics (e.g. TFA, TCA, NaOH, ZnSO4, AcN, MeOH) • Sample prep but NOT an extraction method 160 Accuracy of quality control samples 140 120 • • • • 100 80 60 40 Not selective (relatively dirty extracts) Proteins, peptides (<5000 Da) are not precipitated Additional clean-up (ex. filtration) is needed Not sensitive (sample dilution + high background) 20 0 0 10 20 30 40 50 60 70 80 Number of quality control samples 10 Protein Precipitation Validation experiments Method Development Sample prep LC-MS OK? N Y Sample prep LC-MS Valid? Y SOP N Select new technology • Easy to perform, just mix solvents – centrifuge/filtrate • Not a selective sample clean-up • Relatively high chance on validation & run failures # Ion-suppression # Recovery losses • Method Development and sample prep during day time 11 Liquid/Liquid Extraction Validation experiments Method Development Sample extraction LC-MS OK? Y Sample extraction LC-MS N Valid? Y SOP N Select new conditions Select new technology • Long method development time Limited amount of non-water soluble extraction solvents Examine the influence of pH, ionic strengths, temperature to shift equilibrium. • Good chance on successful validation & run • Sample prep during day time 12 On-line Solid Phase Extraction (SPE) Generic Template Sorbent Screening: Systematic (automated) Method Development Not OK Not OK LC vs. XLC OK Sorbent Screening 500 500µµl 10% M 500µ l 5% M eOH eO H 500µ l 10% l AC 500µ 5% l ACNN Wate r LC vs. XLC Not OK OK Optimize Wash LC vs. XLC Not OK OK Optimize Elution time So r So bent rb 1 So r ent 2 So bent rb 3 So e n t r 4 So bent r 5 So r b ent 6 So bent rbe 7 nt 8 On-line SPE method development: Selective on-line SPE methods in 2-3 hours LC vs. XLC OK Biological Standard 13 Bio-analytical process Method development Method Development Criteria Sample Preparation Running of Samples Data Analysis Protein Liquid-liquid Off-line SPE Precipitation Extraction Simplicity MD time Validation failures Report Column switching On-line SPE ☺ ☺ & $ $ ☺ ☺ $ ☺ $ $ $ $ ☺ ☺ $ /☺ $ /☺ ☺ $ ☺ Method Development: - Overall score ☺=9 $=6 &=3 21 21 24 18 24 14 Bio-analytical process Assay Performance Method Development Sample Preparation Running of Samples Data Analysis Report Assay Performance: • Clean extracts - No ion suppression or enhancement • Reproducibility • Sensitivity 15 Clean extract Why clean extracts? S/N ratio for low concentrations Contaminants LC/MS maintenance Recovery Sensitivity Matrix dependant Ion suppression Accuracy Selectivity Reproducibility Impurities 16 Clean extracts Infusion chromatograms*, SPE plasma, 2.5 min run Protein precipitation Oasis LLE C2 C8 C18 * from Rapid Commun. Mass Spectrom. 13 (1999) 1175-1185 17 Clean extract Infusion chromatogram*, SPE plasma, 20 min run Protein precipitation Oasis LLE C2 C8 C18 * from Rapid Commun. Mass Spectrom. 13 (1999) 1175-1185 18 Clean extract Infusion chromatogram*, 5 injections, 2 min run cumulative effect Protein precipitation Oasis LLE C2 C8 C18 * from Rapid Commun. Mass Spectrom. 13 (1999) 1175-1185 19 Column switching Fixed pre-column different response / performance over time, depends on: • Sample pre-treatment (protein precipitation, filtration, centrifugation is needed before Extraction Column column switching method) • Injection volumes • Number of injections • Pre-column regeneration steps • Pre-treatment steps are needed • Sample preparation time > LC-runtime 20 Off-line SPE: Automation Technology Zymark Rapidtrace Gilson ASPEC liquid handler • Variability in pipetting steps Tomtec Quadra • Off-line SPE vacuum manifolds - differences in sample viscosity • Differences in sample processing flows Zymark Autotrace Beckman Coulter Biomek steps " higher assay variations • Multiple 21 On-line SPE: Automation technology On-line SPE using a disposable extraction cartridge: • • • • • Minimal sample handling steps No sample carry-over from extraction cartridge Positive high-pressure, vacuum manifolds are not used Controlled flow rates Sequential, each sample is prepared within the LC-MS runtime 22 Bio-analytical process Assay Performance Method Development Sample Preparation Criteria - Clean extracts - Reproducibility - Sensitivity Running of Samples Data Analysis Protein Liquid-liquid Off-line SPE Precipitation Extraction Report Column Switching On-line SPE $ $ & $ & ☺ ☺ $ ☺ $ $ ☺ ☺ ☺ ☺ & $/ & $/ ☺ $ ☺ 15 18 24 21 27 Assay Performance - Overall score ☺=9 $=6 &=3 23 Bio-analytical process Workflow Method Development Sample Preparation Running of Samples Data Analysis Report Workflow / sample throughput: • Sample pre-treatment • Automation • Integration with MS software: - Traceability - CFR 21 Part 11 compliancy 24 Sample pre-treatment steps Aspects of manual handling steps: • • • • • Exposure to samples/chemicals Human errors Operator variability During daytime only Tracking and traceability issues 25 Sample pre-treatment steps Aspects of automated steps: # Instrument variability # Several sample manipulation steps # Learning curve to make the automation work # Human errors during transfer steps # Tracking – traceability issues # During daytime only, often not truly a “walk-away” automation 26 Off-line SPE Manual step Retrieve samples To 96-well plate Add IS Add Buffer Automated step Unattended step Elution Extraction Vortex Dry-down Reconstitute To LC/MS Transfer step • Many transfer steps • Automated – semi automated process • Sample extraction during day time 27 On-line SPE Manual step Automated step Retrieve samples To 96-well plate SPE-LC/MS Unattended step Transfer step • On-line IS addition • Minimal transfer steps • Integrated automation • Sample extraction day & night 28 The Broken Link File Lab journal Retrieve info Prep Method Tracing Database LIMS Sample manipulation steps: Searching….. • xdirect minuteslink of vortexing ' No between sample prep • y minutes centrifuging at z r.p.m. ' method and LC-MS method • p µL of sample in vial q to position A1 ' • a ml/min conditioning with b% of solvent c ' • d ml/min loading with e% solvent f ' 29 On-line SPE: integrated automation Aspects of integrated automation: • Minimum sample manipulation - transfer steps • Less chance on human errors • Day & night automation • Tracking - traceability guaranteed 30 Information flow, integrated automation Sample List On-line SPE/MS LC/MS software software LC/MS method On-line SPE/MS method Results &&Results Sample prep Method Benefits of an integrated information flow: • Sample prep data linked with LC-MS data • Reduced chance of errors • Easy tracing • 21CFR-11 compliant 31 Bio-analytical process Workflow Method Development Sample Preparation Criteria - Sample pre-treatment - Automation - MS data integration Running of Samples Data Analysis Protein Liquid-liquid Off-line SPE Precipitation Extraction Report Column Switching On-line SPE ☺ ☺ & ☺ & & ☺ $ & $ ☺ $ $ ☺ ☺ $/☺ & $ $/☺ ☺ 21 24 Workflow: - Overall score ☺=9 $=6 &=3 21 15 18 32 Bio-analytical process Overall process Method Development Sample Preparation Running of Samples Data Analysis Report Method Development Sample Preparation Running of Samples Data Analysis Report Method Development Sample Preparation Running of Samples Data Analysis Report Overall Process: Protein Liquid-liquid Off-line SPE Precipitation Extraction Column Switching On-line SPE - Method Development - Assay Performance - Workflow $/ ☺ & $/ ☺ $/ ☺ $/ & & ☺ $/ ☺ $ $ $ $/ ☺ ☺ ☺ ☺ - Overall score &/ $ & $/ ☺ $ ☺ 19 18 22 20 25 33 Summary • • Each sample prep technique has its strong and weak points to consider However, before you select a sample prep technique examine the performance of the sample preparation method in the entire bio-analytical process: Method Development, Assay Performance, Workflow Assay Performance Method Development • Often only the method development time is the main criterion to select the sample prep technique. (changing “Quick and Dirty” sample prep methods to “Quick and Clean” methods will give more robust assays and a higher workflow) • When the sample prep technique in automated systems is integrated with the LC-MS data it will be easier to be compliant with the CFR21 part 11 regulations, it will save you reporting time and this will result in shorter project cycle times Workflow 34 Technology Leader in automated Front-end Sample Handling n o i s s u c s i D – s n o i t s e ? ? u n o i Q t a t presen nd.com the ll a f o o h k y r p a sp Co @ k a a d.h r a r e g : E-mail 35