Alberta Agriculture Pictures

Alberta Agriculture Pictures
in association with
The Lab Experiment That Ate My Brain Films
presents
1
Scary Reality #1
• LC-MS/MS has led to big expectations in food
safety and environmental analysis
o multiresidue or multiclass residue methods
o minimal cleanup required with LC-MS/MS due to:
- high specificity
- high sensitivity
• labs under pressure to perform more testing:
o larger number of samples
o larger number of analytes
Scary Reality #2
• development of multiresidue LC-MS/MS methods has
led many scientists down torturous path to insanity
o matrix effects frequently encountered
o most co-extracted compounds are “invisible” in MRM
MS/MS
2
More Scary Realities
• matrix effects independent of mass analyzer system
o observed in single quad, triple quad, ToF
• matrix effects dependent upon:
o analyte
o nature of co-eluting components
o concentration of co-eluting components
• matrix effects can be huge pain in neck
o quantitation can be unreliable
Coping with Matrix Effects
1.
matrix matched standards
o variability in matrices can cause problems
2.
use of internal standards
o limited success with analogous compounds
o few isotopically-labeled compounds available
3.
quantitation using method of standard addition
o can become very time-consuming
3
Options for Minimizing Matrix Effects
1.
2.
3.
selective extraction
selective cleanup
chromatographic separation of interfering
components
•
all 3 options become increasingly
difficult as # analytes ↑
method development becomes a
nightmare
•
Creating Our Own Special Little Monster
• screening for sulfamethazine in hog
urine
• program in place since mid-1990s
• not providing accurate snapshot of
current state of industry
o SMZ use has declined
o not sampling sows
• replace with screening of antibiotic
residues in pork kidney
o multiple compounds from different
classes
4
erythromycin
lincomycin
tetracycline
penicillin G
trimethoprim
sulfamethazine
First Attempt at Building the Monster
• need to use suitable extraction solvent to permit recovery of all
analytes
• avoided aqueous buffers due to pH incompatibilities
o acidic or basic conditions not good for macrolides
o tetracyclines usually extracted with McIlvaine buffer (pH = 4)
• logical choice: organic solvent
o MeOH:ACN (1:1)
5
First Attempt at Building the Monster
Homogenized Tissue
2.5 g extracted with 5 mL MeOH:ACN
• inject small quantity of relatively dilute
extract
o helps reduce matrix effects
• divert flow from MS except during
analyte retention window
• able to see all analytes at 1/10 of
MRL
LC Analysis
2 uL injection volume
0.1% FA in water, ACN
MS/MS Analysis
ESI +ve
2 MRM per analyte
First Monster
•
•
•
•
initial results looked promising
good recoveries and reproducibility
no sample cleanup
method performance degraded after
large number of extracts injected
o chromatographic peak shape
deteriorated
o significant ionization suppression
observed for some analytes
6
What Happened and What to Do About It?!
• time to rack your brain:
o need to understand
nature of matrix
o what sort of components
are likely to be coextracted?
Hope the boss is understanding!
Curse of the Phospholipids
• large quantities of lipids co-extracted with organic solvent
• phospholipids known to cause significant matrix effects
o ionization suppression
• problems became progressively worse with more injections on LC
phosphatidylcholines (PCs)
lysophosphatidylcholines (LPCs)
(R = C14 to C24 alkyl group with 0 to 4 double bonds)
7
Minimizing Matrix Effects – The Nightmare Continues
1.
selective extraction
o not an option
o switched to 100% ACN
I’m baacckk!!
- phospholipids slightly less soluble in ACN
2.
selective cleanup
o may be possible but likely difficult
3.
chromatographic separation of interfering
components
o relatively easy in this case
Building a Better Monster – LC Considerations
8
beta lactams
lincosamides + macrolides
sulfonamides + trimethoprim
tetracyclines
Building a Better Monster – Cleanup Considerations
• large quantities of phospholipids being
injected into instrument
o relying heavily on LC separation
o potential for contamination of LC injector
and column
o can we at least reduce amount of
phospholipids and other potential
interferences??
9
Generic SPE
• many cleanup methods in literature use SPE to selectively retain
analytes
o interferences removed with wash
o analytes subsequently removed with organic solvent
• won’t remove PCs and LPCs with this approach
• need to consider chemistry of:
o phospholipids
o analytes
• most tissue PCs and LPCs with C16, C18, and C20 alkyl chains
o 0 to 4 double bonds
• retention behavior of phospholipids on C18 stationary phase
determined by partitioning
o depends mainly on number of C atoms in alkyl chain
o double bonds decrease retention
• should be able to trap some PCs and LPCs with C18
18:0/18:1 PC
16:0 LPC
10
PCs: no cleanup
PCs: dispersive C18 SPE
LPCs: no cleanup
LPCs: dispersive C18 SPE
Second Attempt at Building the Monster
Homogenized Tissue
2.5 g extracted with 5 mL ACN
centrifuge and retain supernatant
Dispersive SPE
shake supernatant with 0.4 g C18
centrifuge and transfer to LC vial
LC Analysis
2 uL injection volume
0.1% FA in water, ACN, MeOH
• biggest change is addition of MeOH
to LC gradient
o ensures complete elution of
phospholipids
o adds 9 min to analytical run
• dispersive SPE adds one step
o easy to process multiple samples
o able to process same number of
samples/day
MS/MS Analysis
ESI +ve
2 MRM per analyte
11
Second Monster
• good recoveries and
reproducibility
• good performance in external PT
• simple sample cleanup
• however:
o still introducing relatively large
quantities of phospholipids into
instrument
o noticed impact on other methods
run on same instrument
Back to the Drawing Board
12
Other SPE Cleanup Options??
Description
Comments
Oasis HLB
• can’t isolate analytes from bulk of phospholipids
silica
•
•
•
•
strong CX
can remove phospholipids from largely organic extract
also removes tetracyclines and lincomycin
PCs and LPCs retained
NH4OH/MeOH doesn’t elute tetracyclines
Things Are Getting Hairy
• what about C18 SPE??
• in order to retain analytes on C18,
need to either:
o evaporate all ACN from extract
- some analytes will elute with even small
percentage of ACN present
- precipitation occurs
- potential loss of analytes??
o dilute extract with very large quantity of
water
- not very practical
13
Popping Out When You Least Expect It
• phospholipids relatively insoluble in
acetone
• dilute ACN extract 8X with acetone
o some precipitation of phospholipids
observed
o supernatant stills contains phospholipids
• elute through C18 SPE and collect
eluant
• easy to evaporate final extract
PCs: no cleanup
PCs: C18 SPE with acetone
LPCs: no cleanup
LPCs: C18 SPE with acetone
14
Future Work
• C18 SPE with acetone looks
promising
?
o remove bulk of PCs + lots of
LPCs
o good recovery of analytes
• need to refine method
• what about other lipids??
o triacylglycerols
- fouling of hardware
What will the monster of the future look like?
Cast
The Monster...........................................................................Tom Thompson
Victor Frankenstein.........................................................................Don Noot
Mad Scientist.................................................................Johan van den Heever
Not Quite So Mad Scientist...........................................................Norine Best
Freddy Krueger........................................................................Desmond Tang
Woman Terrorized by Axe-wielding Boss........................................Joy Komarnicki
Toxic Avenger..................................................................Marlise Siegenthaler
Woman Terrorized by Phospholipid Molecule.................................Renata Limanowka
Terminator............................................................................Chii-Guary Tsai
Invisible Man..............................................................................Chung Lam
Lawrence “The Wolfman” Talbot...................................................Ervin Prommer
Phantom of the Laboratory...............................................Guillermo Recinos-Diaz
15