What You Can’t See Can Hurt You How MS/MS Specificity Can ...

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


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)


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


monensin in raw milk(0.1 ng/mL)


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


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


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


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


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


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


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


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)


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??


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


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


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)


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


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


tetracyclines

sulfon-amides

macrolides


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)


Separation of Ionophores and Phospholipids by HPLC using Silica

ionophores unretained

LPCs

PCs

PEs


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


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


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


A B

C

D

AFLBchickenmuscle

FAPASchickenmuscle

A

B

C

D

LAS

NAR

SAL

NIG

MON


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


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


Questions??

What You Can’t See Can Hurt You How MS/MS Specificity Can ...

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