what you can’t see can hurt you how ms/ms specificity can
TRANSCRIPT
Alberta Agriculture & Food Food Safety Division
What You Can’t See Can Hurt You
How MS/MS Specificity Can Bite Your Backside
Johan van den Heever, Tom Thompson, and Don NootAlberta Agriculture & Food
Food Safety DivisionAgri-Food Laboratories Branch
Alberta Agriculture & Food Food Safety Division
Advances in Trace Organic Residue Analysis
early methods based on GC or HPLC techniques
potential of hyphenated GC-MS or LC-MS recognized but not immediately realized
• vacuum requirements of MS not compatible with GC or LC
GC-MS routinely employed by 1980s• capillary columns permitted direct interfacing of GC to MS
LC-MS routinely employed in 1990s• facilitated by development of atmospheric pressure ionization
interfaces (ESI and APCI)
Alberta Agriculture & Food Food Safety Division
Advances in Trace Organic Residue Analysis
MS with selected ion monitoring provided:• high sensitivity, selectivity and specificity
permitted multiresidue analysis methods
complex matrices may provide interferences in GC-MS or LC-MS
• possible to have co-eluting compounds with parent or fragment ions having same m/z value chosen for SIM
MS/MS with selected reaction monitoring provides reduced chemical noise
• lower probability of precursor/product ions having same combination of m/z values
Alberta Agriculture & Food Food Safety Division
Advantages of MS/MS over MS
low probability different molecules will have same precursor/product ion transition
• peak identification easier• improved sensitivity (better signal:noise)
theoretically:
less extensive cleanup required
less sophisticated chromatographic separation required
Alberta Agriculture & Food Food Safety Division
Reality
ionization ultimately impacts quantitation regardless of whether MS or MS/MS
matrix effects include:• ionization suppression• ionization enhancement
not as commonly observed in GC-MS or GC-MS/MS
• higher chromatographic resolution
• non-volatile, ionic species not eluted
Alberta Agriculture & Food Food Safety Division
LC-MS/MS in Trace Organic Residue Analysis
LC methods applicable to broader range of analytes compared to GC
• compounds with high boiling points• thermally labile compounds• no derivatization required for polar functional groups
dramatic increase in utilization of LC-MS/MS during recent years
matrix effects significant in LC-MS/MS, especially ESI
cleanup and/or chromatography can be critical
Alberta Agriculture & Food Food Safety Division
Ionization Enhancement – Erythromycin in Surface Water
extraction and cleanup using anion exchange SPE + Oasis HLB SPE
both MS/MS transitions for erythromycin are clearly observed
unobserved co-eluting matrix components result in increased signal for surface water extract
recoveries >150% common
50 ppb reagentstandard
surface waterspiked at 50 ppb
Alberta Agriculture & Food Food Safety Division
Matrix Effects
dependent upon:• concentration and nature of co-extracted matrix components• nature of analyte• average recoveries of macrolides in series of spiked surface waters
o tylosin: 79 + 12%o erythromycin: 212 + 53%
matrix-matched standards often improves quantitation• variability in matrix must be minimal
o does not work for surface waters from vastly different sources
isotopically labelled internal standards correct for matrix effects but:
• very expensive• limited availability
Alberta Agriculture & Food Food Safety Division
Overcoming Matrix Effects
best options:1. selective extraction and/or cleanup2. chromatographic separation of interferences
most co-extracted compounds are transparent in MS/MS
need to understand nature of sample matrix to successfully employ options 1 and/or 2
Alberta Agriculture & Food Food Safety Division
Overcoming Matrix Effects
selective extraction and/or cleanup• possible when analyzing compounds from same family with
similar properties (e.g., tetracyclines)
chromatographic separation of interferences• may be possible for small number of analytes
both options more difficult when analyzing large number of compounds from different families• tetracyclines, sulfonamides, β-lactams, fluoroquinolones in eggs
by SPE and LC-MS/MS (Heller et al., 2006)• often must seek compromise
Alberta Agriculture & Food Food Safety Division
Multiresidue Antibiotics in Tissue
simultaneous determination of:
• sulfonamides• tetracyclines• macrolides
generic SPE cleanup
employed for kidney or muscle tissue
Analysis by LC-ESI-MS/MS
SPE Cleanup with Oasis HLB
Homogenized Tissue(extract with ACN)
Alberta Agriculture & Food Food Safety Division
40 ppb
100 ppb
150 ppb
200 ppb
Tilmicosin in Swine Kidney
In successive injections of more concentrated matrix-matched standards, signal initially increases & then decreases
Cause of signal suppression??
Alberta Agriculture & Food Food Safety Division
50 ppb
Sulfadoxine + Sulfadimethoxinein Swine Kidney
100 ppb
75 ppb
20 ppb
Slow loss of sensitivity observed for sulfadoxine with successive injections
Rapid loss of sensitivity observed for sulfadimethoxine with successive injections
SDX and SDMX are isomers with same MRMs
Alberta Agriculture & Food Food Safety Division
Co-extracted Matrix Components
ACN effectively extracts various classes of antibiotics from homogenized tissue
• potentially extracts much more
resulting extracts are coloured prior to SPE
SPE does remove some colour
evaporation of extract to dryness reveals oily residue even after SPE
Alberta Agriculture & Food Food Safety Division
Co-extracted Matrix Components
ACN also effectively extracts phospholipids (known to cause ionization suppression)
N+ O
PO
O
OO
O
R2
O
R1O
N+ O
PO
O
OHO
O
R2O
OP
O
O
OO
O
R2
O
R1
N+
H
HH
O
phosphatidylcholine (PC) lysophosphatidylcholine (LPC)
phosphatidylethanolamine (PE)
R1, R2 = alkyl chains(mainly C14 to C24 with 0 to 4 DB)
Alberta Agriculture & Food Food Safety Division
Cause of Ionization Suppression
co-extracted materials not completely eluted from LC column
components remaining on column from previous injection(s) may elute during subsequent analyses
previous gradient elution program with 0.1% FA in water and ACN
• total run time = 27 min
included 0.1% FA in MeOH with extended wash to ensure complete elution of PLs
• new total run time = 36 min
Bottom line: ionization suppression eliminated
Alberta Agriculture & Food Food Safety Division
Monitoring for Phospholipids in Tissue Extracts by Parent Scan MS/MS
N+ O
PO
O
OHO
O
R2O
+H
N+ O
PO
O
OO
O
R2
O
R1O
+2H
Alberta Agriculture & Food Food Safety Division
Ionophores in Chicken Muscle
method under development
co-extracted phospholipids not separated from ionophores using Oasis HLB or C18 SPE
• retention of PLs based on longchain alkyl groups
can be removed by silica SPE• retention based on polar
head group of PLs
ionophores not retained by silica SPE
Analysis by LC-ESI-MS/MS
SPE Cleanup with Silica
Homogenized Tissue(extract with ACN)
Alberta Agriculture & Food Food Safety Division
Separation of Ionophores and Phospholipids by HPLC using Silica
ionophores unretained
LPCs
PCs
PEs
Alberta Agriculture & Food Food Safety Division
Analysis of Ionophores in External PT Chicken Muscle
ave response of ISTD for matrix-matched calibration standards:
411,026 (%RSD = 4.8, n = 8)
ISTD response for replicate FAPAS samples:sample A: 232,984sample B: 258,590
ISTD response for replicate FAPAS samples spiked at 36 ppb (post extraction):
spiked sample A: 238,655spiked sample B: 259,856
Alberta Agriculture & Food Food Safety Division
Comparison of Results: External vsInternal Standard Quantitation
Lasalocid Monensin Narasin Salinomycin
tissue A 12 21 0 0 0 0 18 32
tissue B 16 25 0 0 0 0 22 35
spiked tissue A 58 96 36 60 41 71 60 102conc. (ppb)
spiked tissue B 60 92 34 52 39 61 61 96
spiked tissue A 127 208 100 167 114 197 117 194%R of spike
spiked tissue B 122 197 94 144 108 169 108 169
Alberta Agriculture & Food Food Safety Division
Ionophores in Chicken Muscle Extracts
removal of phospholipids using silica SPE verified using LC-MS/MS
difference in ISTD response between matrix-matched calibrators and samples raises flag
What causes difference in response??
Compare extracts using fullscan MS
Alberta Agriculture & Food Food Safety Division
A B
C
D
AFLBchickenmuscle
FAPASchickenmuscle
A
B
C
D
LAS
NAR
SAL
NIG
MON
Alberta Agriculture & Food Food Safety Division
Co-eluting Compounds
fullscan MS suggests series of homologs with alkyl chain(s)
• base peaks differing by 28 Da (C2H4addition)
neutral lipids (triacylglycerols) not removed by silica SPE
interpretation of ESI-MS and ESI-MS/MS spectra difficult
• precursor and product ions of acylglycerols highly dependent upon degree of unsaturation and location of double bonds
OO
R2
O
O
R1
R3
O
O
R1, R2,R3 = alkyl chains(mainly C14 to C24
with 0 to 4 DB)
triacylglycerols
Alberta Agriculture & Food Food Safety Division
Conclusions
specificity of LC-MS/MS can lead to false sense of confidence in method
matrix effects not always corrected by use of:• analogous compounds for ISTDs• matrix-matched calibration standards
specific identify of interference(s) not always known or easily determined
no substitute for thorough method validation
participation in external PT or interlab comparisons invaluable