Automated Sample Preparation. Andrew Duggan, PhD Aman Sharma Shimadzu Scientific Australia

Transcription

1 Automated Sample Preparation Andrew Duggan, PhD Aman Sharma Shimadzu Scientific Australia

2 Complete Workflow Solution Extrahera Analyte Extraction Solvent Evaporation Turbovap Analyte analysis LC/MS & GC/MS 2

3 The Shimadzu-Biotage Connection? Brief Overview Biotage & Shimadzu Global collaboration Simply makes sense Biotage = synthesis, sample preparation Shimadzu = purification, analysis Together = Complete Solution Shimadzu Australia Biotage distributor 2016 Benefits for the customer: Exceptional support & service Applications development Reduced costs

4 Brief Overview of Biotage Uppsala Charlotte Corporate headquarter Lund Tokyo / Osaka Cardiff Est ~300 employees ~USD 80m net sales NASDAQ Shanghai

5 Presentation Overview Sample Preparation Automation What is sample prep & why are we doing it? Defining the Goals of Sample Prep Complete workflow solutions Why should we Automate? Extrahera: Features & Benefits Applications & Method Creation Wrap up

6 What is Sample Preparation? The way in which a sample is treated prior to its analysis Sample Matrix Eluent LCMS/GCMS Analysis Interferences That Cause Ion Suppression: Proteins, phospholipids, lysophospholipids, Ionizable Salts Analytes Confirmation & Quantification

7 What is Sample Preparation? The way in which a sample is treated prior to its analysis Process where: Analytes in the sample matrix Transformed so they are suitable for analysis Provides an extract that is: Representative Reproducible Homogenous Clean Appropriate for Instrument analysis

8 What is Sample Preparation? Representative workflow Sample Matrix Eluent LCMS/GCMS Analysis Extraction Purification Analysis Methanol Isopropanol/ Imidazole Aqueous Buffers Ionic liquids etc. LLE SPE SLE PLD PPT QuEChERs Etc. GC/MS GC/MS/MS LC/MS LC/MS/MS Immunoassay 2D HPLC HPLC-FLD CE CE/MS etc.

9 Goals of Sample Preparation Obtain a clean, concentrated sample - appropriate for analysis Clean-up Remove components from sample matrix that may interfere with compounds of interest Reduce system maintenance and down-time Improve chromatography & reproducibility Minimize ion suppression / enhancement issues Concentration Improve sensitivity / increase LOD Solvent Switching Organic solvent to aqueous LC/MS

10 Why should we Automate Sample Prep? Chromatographic Analysis Time Breakdown Sample Preparation & Extraction Sample Analysis (LC/GC) 27% Sample Collection Data Processing Management Automation becomes an exciting proposition when considering the benefits 6% 61% 6% Source of Errors 30% 4% 11% Contamination Columns 19% Operator Sample Introduction 9% 8% 6% 7% 6% Chromatography Integration Instrument Calibration Sample Processing» R.E. Majors, LCGC 9(1), (1991).

11 Why should we Automate Sample Prep? Manual Sample Preparation Labour intensive & expensive Expensive legacy hardware Variations in results from run-to-run Human Error Require rerun samples Additional Lab Techs on demand Result = Costly problems Implementation of Automation Offer advantages & improvements Lab efficiency Data Quality Reduces time per sample Lower cost per sample Well designed automation can balance ROI Extrahera

12 Complete Workflow Solution Instruments Consumables Application support Service expertise 12

13 Complete Workflow Solution Analyte Extraction Solvent Evaporation Analyte analysis Automated Rapid Reproducible High Recovery Selective Robust Rapid Efficient Reliable Accurate Fast Sensitive Extrahera Consumables Turbovap LC/MS & GC/MS 13

14 Automation Biotage Extrahera 14

15 Extrahera Brief Overview Fully Automated High Throughput Positive Pressure Multi-format Touch Screen operation Intelligent pipette use Plates & Columns Extrahera

16 Sample Prep Automation Lets see this in action

17 The Extrahera s Features Automate Sample Preparation Solid Phase Extraction Supported Liquid Extraction Protein Precipitation Phospholipid Depletion Simple routine operation Intuitive software Easy setup of methods Extrahera Small Footprint Compact two tier design Fully enclosed fume hood Allows on bench installation Accommodates lab growth Flexible plate & cartridge processing Customizable specifications

18 Designed to Improve Efficiency Directly lowers cost per sample Redeployment of skilled analysts Reduces reruns through errors Simple easy-to-use software Touch-screen function Intuitive Simple menu driven operation Fast method development Audit trail Intelligent hardware design Builds confidence & adaptability Multi-format capability Safe & secure SLE+ & SPE plates & Columns

19 Providing Optimum Data Quality Positive pressure Provides uniform processing of each sample, regardless of viscosity Pre-configured methods Samples are prepared & processed in exactly the same way every time Eliminating human & system variations Allows easy development of new method parameters Maximum precision & accuracy Obtained through precise control of each parameter, including air gaps, aspiration & dispense speeds, & movement of the pipetting head

20 Automation Biotage Extrahera Components 20

21 Software 12 touch screen No PC required Report and method export USB connectivity LAN connectivity

22 Positive Pressure Processing Samples processed through positive pressure dual flow heads Consistent flow Across entire plate Even slow flowing or viscous samples Two operation modes Low Flow 0 to 10 ml/min High Flow 600 ml/min SPE or SLE+ applications Need contact time of sample with media to ensure recovery = Low flow SPE methods Require complete media dryness or solvent miscibility issues = High flow

23 Interchangeable Heads Designed to enable processing of 96-well plates or columns from one common platform Simple and quick interchange of heads

24 Intelligent Liquid Handling Utilizes 1000uL disposable pipette tips Two sets of tips Sample Disposable Reagent Optional tip reuse Aspirate/Dispense Conditions Fully customisable Five solvent on-demand reservoirs Five static solvent reservoirs Accurate from 50 to 1000 ul 50 μl: ±2.0% accuracy and 1.0% CV 100 μl: ±2.0% accuracy and 1.0% CV 500 μl: ±1.5% accuracy and 1.0% CV 1000 μl: ±1.0% accuracy and 1.0% CV

25 Waste Removal Space saving two tier, carousel design Flow through plate, Minimizing cross-contamination & cross-talk Three elution positions

26 Method Development Options Protein Precipitation (PPT+) Phospholipid Depletion (PLD+) Supported Liquid Extraction (SLE+) Solid Phase Extraction (SPE) 26

27 Method Development Considerations Workflow & Cost Sorbent selectivity & Matrix effects Analytical instrument sensitivity & cost to maintain Sorbents: Polar or Non-polar or Inert Acidic, Basic or Neutral PPT+, PLD+, SLE+, SPE, MIPS Format and Throughput: Columns 1, 3, 6 ml & larger 96-well plates Plates & Columns

28 The Options Workflow & Cost Workflow Products Cost Low Protein precipitation (PPT+) Minimum 2 workflow steps. Lower Number of Steps Phospholipid depletion (PLD+) Minimum 2 workflow steps. Time Required Complexity Supported Liquid Extraction (SLE+) Minimum 3 workflow steps. More Evolute Express (SPE) Minimum 3 workflow steps. Isolute (SPE) Minimum 5 workflow steps. Higher

29 The Options - Selectivity & Matrix Selectivity Products Matrix Effects Non- Selective Dilute & Shoot Protein precipitation (PPT+) High Phospholipid depletion (PLD+) Supported Liquid extraction (SLE+) Highly Selective Solid-phase extraction (SPE) Non-polar Mixed mode Ion exchange Molecularly Imprinted Polymers Low

30 Option #1 Protein Precipitation (PPT+) 30

31 Protein Precipitation Isolute PPT+ Dispense crash solvent Dispense sample matrix e.g. plasma, blood Apply positive pressure Mix as required Lower frit prevents dripping Collect purified analytes Precipitated proteins retained by depth filter

32 Option #2 Phospholipid Depletion (PLD+) 32

33 Phospholipid Depletion Isolute PLD+ Dispense crash solvent Dispense sample matrix e.g. plasma, blood Apply positive pressure Mix as required Precipitated proteins retained by depth filter Phospholipids retained Depth filter reduces clogging & removes proteins Sorbent layer- removes phospholipids Collect purified analytes Lower frit prevents dripping 33

34 Creating PPT/PLD Methods

35 Creating PPT/PLD Methods

36 Time to relax and have a break!

37 Option #3 Supported Liquid Extraction (SLE+) 37

38 Supported Liquid Extraction Overview Analogous to LLE on an inert support material No true chemical interaction with material No flow through during load Appropriate for biological media Cleaner extracts No emulsion formation Fast load-wait-elute procedure High analyte recovery 38

39 Supported Liquid Extraction Mode of Action Small surface area particles Sample disperses over the surface of the inert material Analytes come into contact with fresh solvent as the organic phase travels through the bed Mimicking a repeat LLE mechanism Large effective surface area at the extraction interface Leads to a very efficient extraction 39

40 Supported Liquid Extraction Isolute SLE+ Pulse of pressure Organic elution solvent added Analytes partition into elution solvent and are collected Aqueous sample loaded onto sorbent bed Aqueous sample flows onto extraction bed and is dispersed in small droplets

41 Supported Liquid Extraction (SLE+) Method Development 41

42 Analyte Properties for Effective Partitioning Maximum amount of un-ionized groups of analyte Ionization affects polarity Distribution Coefficient, Log D Ratio of concentrations of all species Higher = Greater hydrophobicity ph control ph = pka 50% drug ionized 50% neutral 2 ph units below an acid = 99.5% neutral 2 ph units above a base = 99.5% neutral Solubility: Not always known

43 SLE+ Method Selection 43

44 Supported Liquid Extraction (SLE+) Extract Cleanliness Proteins Phospholipids Lysophospholipids 44

45 Protein Analysis Utilizing Gel Electrophoresis ACN Crash Ratio for PPT+ Serum Pre-treated with H 2 O Extracted with MTBE Raw Serum SLE+ Mol. Wt Marker PPT+ = 99% protein removal at 1:3 crash ratio SLE+ = FULL protein removal

46 Phospholipid & Lysophospholipids Comparison of various extraction conditions, PPT+ v. SLE+ Plasma extracted with PPT+ Pre-treatment 1:1 0.5M NH 4 OH Extraction solvent MTBE Pre-treatment 1:1 1% formic acid Extraction solvent DCM Pre-treatment 1:1 1% formic acid Extraction solvent EtOAc 100 2: MRM of 11 Channels ES TIC e7 2: MRM of 11 Channels ES+ TIC 5.63e : MRM of 11 Channels ES+ TIC 5.63e : MRM of 11 Channels ES+ TIC 5.63e7 % Phospholipids % % % : MRM of 5 Channels ES+ TIC 3.27e : MRM of 5 Channels ES+ TIC 3.27e : MRM of 5 Channels ES+ TIC 3.27e : MRM of 5 Channels ES+ TIC 3.27e7 % Lyso phospholipids % % % Time Time Time Time

47 Biotage SLE+ - World Best! Don t take our word for it ask our customers The SLE+ is great. Out performs any other SLE on the market. Am very happy. Brian Cooke, Pathwest, WA 47

48 Supported Liquid Extraction (SLE+) Automation Setup 48

49 Creating SLE+ Methods

50 Creating SLE+ Methods

51 Creating SLE+ Methods

52 Option #4 Solid Phase Extraction (SPE) Isolute Silica based Evolute Polymer based 52

53 Solid Phase Extraction (SPE) Most specific / targeted Can be optimized for groups of compounds & sample matrices Improved sensitivity / increased LOD Requires method development Reduces solvent consumption Requires trained staff

54 #1 Isolute SPE Products Silica based Columns & plates Wide variety of silica based sorbents Reversed Phase Normal Phase Mixed Mode (IEX) Specialty Phases Myco/O&G/Na2SO4/Florisil

55 ISOLUTE SPE Silica based SPE Elution solvent added Analytes are retained Methanol wash Mobile phase equilibration Sample introduced Interferences flow to waste Purified analytes collected 55

56 #2 Evolute Express SPE Products Polymer Based - PS-DVB (ph = 1-14) Columns & plates 5 Phases ABN = Simultaneous extraction of acidic, basic and/or neutral analytes CX = extraction of basic analytes WCX = extraction of strongly basic analytes AX = extraction of acidic analytes WAX = extraction of strongly acidic analytes Excellent lot-to-lot reproducibility

57 EVOLUTE Express SPE Polymer based SPE Wash Elution solvent added Bottom frit prevents dripping Analytes are retained Interferences flow to waste Collect purified analytes 57

58 EVOLUTE Express Load-Wash-Elute SPE Columns & Plates Fast & Efficient Flow Rates Direct loading onto a dry column 58

59 EVOLUTE Express Load-Wash-Elute SPE Columns & Plates Reproducibility is key Extremely consistent results 59

60 EVOLUTE Express Load-Wash-Elute SPE Columns & Plates Load-Wash-Elute Procedure No conditioning or equilibration necessary 60

61 Creating SPE Methods

62 Creating SPE Methods

63 Creating SPE Methods

64 Creating SPE Methods

65 Prepare your Run Tracking Info Solvent Tips Sample Tips Extraction media plate or columns Solvent Reservoirs Select samples to run Method details Carousel LIMs Input

66 Extrahera Summary Dedicated automation platform User-friendly interface Easy to set up, operate & maintain Operates consistently across a wide variety of sample prep techniques & reduces errors Reduces processing time Extrahera is designed to be flexible to meet your changing lab or method needs Saving time & money

67 Evaporation Sample Concentration Solvent exchange 67

68 Evaporation TurboVap 96, II and LV Gas vortex shearing technology End-point detection (TurboVap II Only) Water bath heating source Parallel processing for increased sample throughput SPE Dry well format Single or dual plate option Heated gas flow from below and above

69 Q&A Many thanks for your attention

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