What You Can t See Can Hurt You How MS/MS Specificity Can Bite Your Backside Johan van den Heever, Tom Thompson, and Don Noot Agri-Food Laboratories Branch
Advances in Trace rganic 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 rganic 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 rganic 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 + asis HLB SPE 50 ppb reagent standard both MS/MS transitions for erythromycin are clearly observed unobserved co-eluting matrix components result in increased signal for surface water extract surface water spiked at 50 ppb recoveries >150% common
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
vercoming Matrix Effects best options: 1. selective extraction and/or cleanup 2. 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
vercoming 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 Homogenized Tissue (extract with ACN) SPE Cleanup with asis HLB employed for kidney or muscle tissue Analysis by LC-ESI-MS/MS
Tilmicosin in Swine Kidney 40 ppb In successive injections of more concentrated matrixmatched standards, signal initially increases & then decreases 100 ppb Cause of signal suppression?? 150 ppb 200 ppb
Sulfadoxine + Sulfadimethoxine in Swine Kidney 20 ppb Slow loss of sensitivity observed for sulfadoxine with successive injections 50 ppb SDX and SDMX are isomers with same MRMs 75 ppb 100 ppb Rapid loss of sensitivity observed for sulfadimethoxine with successive injections
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) R1 H N + P R2 N + P R2 phosphatidylcholine (PC) lysophosphatidylcholine (LPC) R1 R1, R2 = alkyl chains (mainly C14 to C24 with 0 to 4 DB) H H N + H P R2 phosphatidylethanolamine (PE)
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 MeH 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 R1 N + P R2 +2H +H H N + P R2
tetracyclines sulfonamides macrolides
Ionophores in Chicken Muscle method under development co-extracted phospholipids not separated from ionophores using asis 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 Homogenized Tissue (extract with ACN) SPE Cleanup with Silica Analysis by LC-ESI-MS/MS ionophores not retained by silica SPE
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: 232,984 sample B: 258,590 ISTD response for replicate FAPAS samples spiked at 36 ppb (post extraction): spiked sample A: 238,655 spiked sample B: 259,856
Comparison of Results: External vs Internal Standard Quantitation Lasalocid Monensin Narasin Salinomycin tissue A 12 21 0 0 0 0 18 32 conc. (ppb) tissue B 16 25 0 0 0 0 22 35 spiked tissue A 58 96 36 60 41 71 60 102 spiked tissue B 60 92 34 52 39 61 61 96 %R of spike spiked tissue A 127 208 100 167 114 197 117 194 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
NAR D SAL AFLB chicken muscle C NIG C B MN FAPAS chicken muscle LAS A B D A
Co-eluting Compounds fullscan MS suggests series of homologs with alkyl chain(s) triacylglycerols base peaks differing by 28 Da (C 2 H 4 addition) R1 neutral lipids (triacylglycerols) not removed by silica SPE R2 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 R3 R1, R2,R3 = alkyl chains (mainly C14 to C24 with 0 to 4 DB)
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??