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Introduction LC-MS/MS Analysis - EURL | Pesticides

Analysis of Amitraz (sum) in samples with incurred residues - Comparison of the approach covering the individual metabolites via LC-MS/MS with the approach involving cleavage to DMA. Julia Hepperle, Irina Sigalov, Dorothea Mack, Sigrid Sch ler, Michelangelo Anastassiades E-Mail: Introduction LC-MS/MS Analysis Amitraz m/z 294/122. The current MRL residue definition (RD) for amitraz Column: Phenomenex Synergi Hydro RP ( m, x 100 mm) Guard-column: Phenomenex AQ C18 (4 x mm). ( Sum of amitraz plus all its metabolites containing the Intensity, cps Mobile Phase: 2,4-dimethylanilin moiety ) requires methods that A: 5 mmol NH4-Formate in water + 5% methanol involve hydrolysis to 2,4-dimethylanilin (DMA). To B: 5 mmol NH4-Formatein methanol Time, min Flow: mL min-1 calculate total amitraz from DMA a 1:2 stoichiometry DMF m/z 150/107.

In 2007 the EURL-SRM distributed a QuEChERS-based method where amitraz parent and its most important MRM -amenable metabolites containing the Analysis of “Amitraz (sum)” in samples with incurred residues -

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Transcription of Introduction LC-MS/MS Analysis - EURL | Pesticides

1 Analysis of Amitraz (sum) in samples with incurred residues - Comparison of the approach covering the individual metabolites via LC-MS/MS with the approach involving cleavage to DMA. Julia Hepperle, Irina Sigalov, Dorothea Mack, Sigrid Sch ler, Michelangelo Anastassiades E-Mail: Introduction LC-MS/MS Analysis Amitraz m/z 294/122. The current MRL residue definition (RD) for amitraz Column: Phenomenex Synergi Hydro RP ( m, x 100 mm) Guard-column: Phenomenex AQ C18 (4 x mm). ( Sum of amitraz plus all its metabolites containing the Intensity, cps Mobile Phase: 2,4-dimethylanilin moiety ) requires methods that A: 5 mmol NH4-Formate in water + 5% methanol involve hydrolysis to 2,4-dimethylanilin (DMA). To B: 5 mmol NH4-Formatein methanol Time, min Flow: mL min-1 calculate total amitraz from DMA a 1:2 stoichiometry DMF m/z 150/107.

2 Was typically assumed reflecting the presence of two Mass transitions used for quantification: Intensity, cps 294 122 (amitraz) DMA moieties in the amitraz molecule. The strong 150 107 (DMF). hydrolysis conditions required and the difficulties in the 163 122 (DMPF). Time, min Analysis of DMA complicate the analytical methods 122 107 (DMA) DMPF m/z 163/122. 128 110 (DMA D6) In-source- fragm. based on this principle. Analysis of amitraz (sum) via DMPF. of Amitraz Intensity, cps DMA is thus rarely conducted routinely by residue Chromatographic separation of amitraz, DMF, control labs. DMPF and DMA was essential to avoid Time, min interferences caused by the in-source DMA m/z 122/107. CH3 CH3 CH3 In-source fragm. fragmentation of amitraz into DMPF and of of DMF 2,4 -DMA. Intensity, cps N N N In-source fragm. DMF and DMPF into DMA.

3 Of DMPF. H3C CH3 Amitraz Time, min CH3 CH3 NH2. Experiments and Results Amitraz, DMA, DMF, DMPF ( g/mL). spiked on QuEChERS extracts of blank pears CH3. H3C NH. 11 pear samples with amitraz treatment history were H3C NH. N O. analyzed using both approaches: (I) QuEChERS. H CH3 CH3 followed by direct LC-MS/MS Analysis of amitraz, DMPF DMF 2,4-Dimethylanilin DMF, DMPF and DMA; (II) QuEChERS followed by hydrolysis and Analysis of DMA by LC-MS/MS . In 2007 the EURL-SRM distributed a QuEChERS- Using approach (I) only DMPF was detected. When based method where amitraz parent and its most mathematically converting the DMPF results to DMA. important MRM-amenable metabolites containing the (using 1:1 stoichiometry) the results obtained were DMA-moiety, namely N-2,4-Dimethylphenyl-N'-methyl- comparable to the DMA results obtained following formamidine (DMPF) and N-2,4-Dimethylphenyl- approach (II) involving chemical conversion to DMA.

4 Formamide (DMF) and DMA are analyzed separately (see Figure below). This suggest that for the samples via LC-MS/MS [1]. Many labs follow this MRM- tested and the hydrolysis conditions used, DMPF is approach and calculate amitraz (sum) from there using virtually the only source of DMA. a 1:1 stoichiometry between DMPF and amitraz. Ratio of However, as amitraz degradation pathways are quite Sample DMA calc. from DMPF. vs. complex and branched, calculation of amitraz (sum) No. DMA determined following hydrolysis using the results of the metabolites can be quite tricky. Aim of this study was to compare the results for amitraz 1 2 (sum) obtained by the two approaches using samples 3 with incurred residues. To enable this, a simple 4 5 QuEChERS-based method involving quantitative 6 hydrolysis of amitraz and its metabolites into DMA was 7 mg/kg 8 developed.

5 0 1 2 3 4 5 6 7 8 9 10 11. 9 Sample No. 10 Hydrolysis of Amitraz and Metabolites DMA calculated from DMPF DMA determined following alkaline hydrolysis Average 11 Samples were extracted using the QuEChERS method without d-SPE step. Alkaline hydrolysis to DMA was Summary and Conclusion performed following addition of aqueous NaOH. Amitraz and its metabolites DMF and DMPF were Methanol was also added to prevent separation of successfully hydrolyzed to DMA in QuEChERS. water. Hydrolysis was shown to be complete for extracts from pears. amitraz, DMF and DMPF, whereas DMA proved to be stable during hydrolysis. DMA D6 was used as ISTD. DMPF was the only component detected in pear samples and turned out to be the only DMA-source. Our results support the use of a 1:1 stoichiometry to PA 031. Hydrolysis scheme 1 mL QuEChERS extract calculate amitraz from DMPF.

6 The alternative addition of internal standard approach involving chemical (or mathematic) EPRW 2012. addition of 1 mL MeOH and 4 mL NaOH (5 N). conversion of DMPF to DMA (with 1:1 stoichiometry). and a subsequent calculation of total amitraz based hydrolysis, 3 h at 90 C cool down on a 1:1/2 stoichiometry underestimates total amitraz addition of 4 mL H2SO4 (5 N) filtration step results by a factor of 2. LC-MS/MS injection References [1]


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