188
T.S. Guldberg et al.
Purpose
The aim of this study was to improve the performance of LC-MS methods for
lipohilic marine biotoxins by introducing internal standards as an analytical tool,
which might compensate for response drift, matrix effects and unpredictable events
during the analytical process.
Methods
Synthesized internal standards were designed from the skeletal structure of
phenylbenzimidazole (PBI) by substitution reactions of different functional
groups. Fifteen substances were tested in methods with negative and positive
ionization mode. Two of the PBIs were further examined in the positive method;
1-pentyl-2-phenylbenzimidazole (Pent-PBI) and 1-heptyl-2-phenyl-benzimidazole
(Hep-PBI). The monitored mass transitions were (precursor > fragment): Pent-PBI
m/z 265.2 > 195.1 and Hep-PBI m/z 293.2 > 195.1.
In addition the polycyclic steroid deoxycholic acid (DHO) was tested for
suitability in the method using negative ionization mode. DHO has comparable
chemical and physical properties with the toxins in the diarrhetic shellfish toxins
(DSP) group, and the monitored transition was m/z 391.1 > 345.2.
The impact of introducing an internal standard for reduced matrix effects
was examined in naturally contaminated mussels (Mytilus edulis) and scallops
(Pectinidae). Parallel extractions were carried out in which one group was extracted
with methanol containing internal standard while the other was extracted using
a published procedure (Stobo et al. 2005). Standard addition was performed by
adding a known amount of multi-toxin solution into crude extracts. The quantified
concentrations were compared with matrix-corrected values.
To monitor response drift with or without internal standard during analysis,
calibration curves with at least five concentration levels were analyzed intermittently
during analytical sequences. CRM
1 calibration standards were fortified in methanol.
Calibration curve slopes, peak areas and response ratios were normalized to comparable levels, tested for significance and confidence intervals (CI) were determined.
Analytical method precision, expressed as %RSD, was calculated based on CRMDSP Mus-b (certified control sample) and naturally contaminated shellfish samples.
Chromatography was performed with two different systems; neutral and alkaline
conditions. The methods used for analysis on single quadrupole MS was AOAC
LC-MS method 88.5 2005 (modified) (Stobo et al. 2005), with a Zorbax Eclipse
XDBC18, 4.6 50 mm, 1.8 m column (Agilent), equipped with an in-line 0.2 m
filter (Agilent). For negative ionization mode mobile phase A was 100 % methanol
1 Certified Reference Materials, NRC-CNRC. Exceptions: DTX-1 from Wako, Japan.
T.S. Guldberg et al.
Purpose
The aim of this study was to improve the performance of LC-MS methods for
lipohilic marine biotoxins by introducing internal standards as an analytical tool,
which might compensate for response drift, matrix effects and unpredictable events
during the analytical process.
Methods
Synthesized internal standards were designed from the skeletal structure of
phenylbenzimidazole (PBI) by substitution reactions of different functional
groups. Fifteen substances were tested in methods with negative and positive
ionization mode. Two of the PBIs were further examined in the positive method;
1-pentyl-2-phenylbenzimidazole (Pent-PBI) and 1-heptyl-2-phenyl-benzimidazole
(Hep-PBI). The monitored mass transitions were (precursor > fragment): Pent-PBI
m/z 265.2 > 195.1 and Hep-PBI m/z 293.2 > 195.1.
In addition the polycyclic steroid deoxycholic acid (DHO) was tested for
suitability in the method using negative ionization mode. DHO has comparable
chemical and physical properties with the toxins in the diarrhetic shellfish toxins
(DSP) group, and the monitored transition was m/z 391.1 > 345.2.
The impact of introducing an internal standard for reduced matrix effects
was examined in naturally contaminated mussels (Mytilus edulis) and scallops
(Pectinidae). Parallel extractions were carried out in which one group was extracted
with methanol containing internal standard while the other was extracted using
a published procedure (Stobo et al. 2005). Standard addition was performed by
adding a known amount of multi-toxin solution into crude extracts. The quantified
concentrations were compared with matrix-corrected values.
To monitor response drift with or without internal standard during analysis,
calibration curves with at least five concentration levels were analyzed intermittently
during analytical sequences. CRM
1 calibration standards were fortified in methanol.
Calibration curve slopes, peak areas and response ratios were normalized to comparable levels, tested for significance and confidence intervals (CI) were determined.
Analytical method precision, expressed as %RSD, was calculated based on CRMDSP Mus-b (certified control sample) and naturally contaminated shellfish samples.
Chromatography was performed with two different systems; neutral and alkaline
conditions. The methods used for analysis on single quadrupole MS was AOAC
LC-MS method 88.5 2005 (modified) (Stobo et al. 2005), with a Zorbax Eclipse
XDBC18, 4.6 50 mm, 1.8 m column (Agilent), equipped with an in-line 0.2 m
filter (Agilent). For negative ionization mode mobile phase A was 100 % methanol
1 Certified Reference Materials, NRC-CNRC. Exceptions: DTX-1 from Wako, Japan.