Cleaning up your sample prep processes Julia C. Drees, Ph.D., DABCC Scientific Director—Chemistry Kaiser Permanente Regional Laboratories Berkeley and Richmond, CA Financial disclosures Nothing to disclose Learning Objectives After this presentation you will be able to: List strengths and weaknesses of common sample preparation techniques used for clinical LC‐MS/MS Develop a plan to select the best sample prep automation for your lab Describe pitfalls and solutions encountered when using automated liquid handlers for clinical LC‐ MS/MS sample preparation
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Cleaning up your sample prep processes
Julia C. Drees, Ph.D., DABCC
Scientific Director—Chemistry
Kaiser Permanente Regional Laboratories
Berkeley and Richmond, CA
Financial disclosures
Nothing to disclose
Learning Objectives
After this presentation you will be able to: List strengths and weaknesses of common sample
preparation techniques used for clinical LC‐MS/MS
Develop a plan to select the best sample prep automation for your lab
Describe pitfalls and solutions encountered when using automated liquid handlers for clinical LC‐MS/MS sample preparation
Common Sample Prep Options
Dilution
Protein Crash or Ultrafiltration (PPT)
Liquid‐Liquid extraction (LLE)
Solid Phase extraction (SPE) – can be offline or online
Dilution
Usually for urine drug confirmations
Typically 10‐20x dilution
Add a filtration step for added robustness
Protein precipitation (PPT)
Avoid adding internal standard (IS) to precipitation solvent
Ideally a ratio of 3:1 precipitant:sample
Options to include phospholipid removal for improved clean‐up and robustness
Must optimize time and parameters for:
Mixing (vigorous; vortex or wide‐bore tip)
Centrifuging (need tight pellet) or vacuum/positive pressure (robust enough?)
Liquid‐Liquid Extraction (LLE)
Best for neutral analytes
Ideally 10:1 solvent:sample
Mixture of polar and nonpolar solvents
More polar → more junk
Phase separation with freeze/pour (manual)
Challenging to automate
Use round deep well 96‐well plates
Aggressively stress test for cross‐contamination
Supported liquid extraction (SLE) is easier to automate
Solid Phase Extraction (SPE)
Most chances for using scientific approach for optimization
Ion exchange SPE is most selective (charged analytes)
Wash aggressively
Ideally twice, 2D, using pH changes and organic solvents
Sacrificing recovery in favor of clean samples is OK
Elute with small volume for more concentrated samples and/or quicker dry times Adapted from Russell Grant, Ph.D. – MSACL Quant. MS Development
& Validation short course, with permission
Comparing extraction modes
Rank ordered 1=best, 4 = worst.
* = Ion exchange SPE mode etc.
Table from Russell Grant, Ph.D. – MSACL Quant. MS Development & Validation short course, with permission
Gerstel MPS Workstation with in‐tip dispersive SPE
User‐developed online SPE
Most LC‐MSMS vendors have packages/software ‐extraction pump(s), switching valves, installation, application notes
Automated Liquid Handling (ALH)
Many vendors and style options
Useful even just for tube to plate before online or off‐the‐deck extraction (e.g., positive pressure SPE)
*Most affordable option
Online Extraction vs. Automated Liquid Handling (ALH)Parameter Online ALH
Tech hands on time/sample Less More
LC sophistication required More Less
Flexibility Less More
Suited to many assays Less More
Suited to 1 high‐volume assay More Less
Sensitivity per L starting sampleǂ More Less
Risk of carryover More Less
Risk of cross contamination (96‐well plates) Less More
Throughput Less* More
Adapted from Judy Stone, Ph.D.
ǂIf limited sample available (e.g., pediatrics), online is better. ALH requires dead volume both in sample tube and after reconstitution*Multiplexed online extraction increases throughput
Automated Liquid Handling –Case Histories
Sample prep for 25‐OH Vitamin D analysis by LC‐MSMS
Thanks to my colleagues who helped troubleshoot these cases
Judy Stone, Ph.D.—Center for Advanced Laboratory Medicine, UCSD
Bret Martin – Applications Programmer, Hamilton Robotics
1. Transfer 50 µL IS from reservoir to plate (8 tips, pick up & dispense 12 times)
IS reservoir
2. Transfer serum from tubes to plate (8x12 tips) & mix w tips
Tube to Plate
3. Move plate to 2nd ALH (96 channel head)
4. Transfer serum + IS to PPT/phospholipid removal plate
5. Transfer precipitating reagent to extraction plate and mix with tips
7. Sealed plate on LC-MSMS
Plate Stamping
6. Vacuum to elute into collection plate below
Case 1 –The Touch‐OffTask
Pipet serum and internal standard (IS) to a 96‐well plate
2 precipitation reagents instead of 1 (more robust)
Total volume had to stay the same
IS volume must be decreased to 25 µL
Problem: %CV ↑ to 6‐9% when IS volume ↓ from 50 µL to 25 µL
Re‐measure bottom of plate dimensions w calipers & adjust height of dispense – not fixed
Lower height of dispense (with only 25 µL not touching off on bottom of plate?) – not fixed
Slow speed of dispense – not fixed
Pause after dispense – not fixed
Blow out of residual volume in tips – not fixed
Solution
Dispense serum 1st
Dispense IS 2nd ‐ touch off to serum (liquid) instead of questionable touch off to (dry) plate
Intra‐assay % CV decreased to 2‐4%
Trade offs
Touch off to serum requires new tip each well
Increased tip cost (from 8 to 96 tips)
Tube to plate time increased ~5 min/plate
Lesson learned – small changes can make a big difference in liquid handling precision
2. Transfer 50 25 µL IS from reservoir to plate (8 tips, pick up & dispense 12 times) (8x12 tips & mix)
IS reservoir
1. Transfer serum from tubes to plate (8x12 tips) & mix w tips
Switched order of tube to plate. Increased time and tip usage but superior precision.
Case 2 – Missing filtratesTask
Precipitate serum + IS in a hybrid (protein & phospholipid removal) filtration plate –transfer filtrate to collection plate (vacuum)
Major constraint
Don’t move plates off deck during the process to maintain viable work flow for 25 plates/day
Problem
First few plates look good
Then a few wells in every plate –no filtrate transferred to collection plate
All other wells in those plates have expected recovery
?
Investigation
Change of plate lot? – not fixed
Break through of filtration membrane or quick flow through in surrounding wells causing inadequate vacuum for problem wells? – unable to prove or disprove
Clogged membrane in problem wells –why OK before & not now?
Eureka!
Initial studies used frozen aliquots of pooled serum