16 Searching for Internal Standard for Chemical Routine Analysis. . .
189
while mobile phase B was 100 % aqueous containing 50 mM ammonium acetate.
A gradient from 30 to 90 % A was run over 2 min, and then held for 3 min. At 5 min
the composition was reset to the initial conditions and 2 min equilibration time was
allowed. The flow rate was set at 0.7 ml min
1 and 5 l of each sample was injected
onto the column at 50
ı C.
For analysis in positive ionization mode, mobile phase A was 100 % methanol.
Mobile phase B and C were 100 % aqueous, with 50 mM ammonium acetate and
25 mM formic acid, respectively. Starting with isocratic conditions of 12.5 % of
eluent B and C for 2.5 min, a linear gradient from 75 to 85 % A was run over 3.5 min.
At 6 min the composition was reset to the initial conditions and 2 min equilibration
time was allowed. The flow rate was set at 1.0 ml min
1 and 10 l of each sample
was injected into the column at 30
ı C. The autosampler maintained 4
ı C, and the
detector (Agilent MS-1956B) was equipped with an electrospray source (ESI).
For triple quadrupole analysis, alkaline conditions described in EU harmonised
SOP LIPO LC-MS/MS (EU-Harmonised Standard Operating Procedure for determination of Lipophilic marine biotoxins in molluscs by LC-MS/MS & version 4.
Retrieved July 2011; Gerssen et al. 2009a) were analysed with X-Bridge C18,
2.1 50 mm, 2.5 m column (Waters), carried out on an Agilent MSMS-6460
(1200-series), electrospray ionization combined with positive and negative ionization modes. The mobile phases consisted of H 2 O containing 0.05 % ammonia
(pH 11) in channel A, and 95 % acetonitrile containing 0.05 % ammonia in
channel B. Analysis were performed by running a linear gradient elution, starting
from 10 to 90 % B from 1 to 6.7 min, followed by a 1.3 min hold at 90 % B, a
10 % A, decreasing to 10 % B over 2 min, and holding for 3 min until the next
injection. The flow rate was 0.7 ml min
1 , and the injection volume was 10 l at a
column temperature of 40
ı C. All results are, as far as possible, evaluated without
distinction on instruments or methods.
Results and Discussion
Retention Times
PBI containing short aliphatic chains eluted too early in both ionization modes,
or did not ionize in negative mode at all. Two of the PBI’s; Hep-PBI and PentPBI, were tested further in the positive method, even though they differ chemically
from the lipophilic toxins. Hep-PBI coeluted with azaspirazid-1 (Aza-1) while PentPBI, which is slightly more polar than Hep-PBI, eluted between pectenotoxin-2
(PTX-2) and Aza-1. Because of this, it is reasonable to assume that Hep-PBI will be
appropriate as internal standard for Aza-1. The retention time of DHO was close to
dinophysistoxin-1 (DTX-1), which may indicate DHO as a suitable internal standard
for DTX-1.
189
while mobile phase B was 100 % aqueous containing 50 mM ammonium acetate.
A gradient from 30 to 90 % A was run over 2 min, and then held for 3 min. At 5 min
the composition was reset to the initial conditions and 2 min equilibration time was
allowed. The flow rate was set at 0.7 ml min
1 and 5 l of each sample was injected
onto the column at 50
ı C.
For analysis in positive ionization mode, mobile phase A was 100 % methanol.
Mobile phase B and C were 100 % aqueous, with 50 mM ammonium acetate and
25 mM formic acid, respectively. Starting with isocratic conditions of 12.5 % of
eluent B and C for 2.5 min, a linear gradient from 75 to 85 % A was run over 3.5 min.
At 6 min the composition was reset to the initial conditions and 2 min equilibration
time was allowed. The flow rate was set at 1.0 ml min
1 and 10 l of each sample
was injected into the column at 30
ı C. The autosampler maintained 4
ı C, and the
detector (Agilent MS-1956B) was equipped with an electrospray source (ESI).
For triple quadrupole analysis, alkaline conditions described in EU harmonised
SOP LIPO LC-MS/MS (EU-Harmonised Standard Operating Procedure for determination of Lipophilic marine biotoxins in molluscs by LC-MS/MS & version 4.
Retrieved July 2011; Gerssen et al. 2009a) were analysed with X-Bridge C18,
2.1 50 mm, 2.5 m column (Waters), carried out on an Agilent MSMS-6460
(1200-series), electrospray ionization combined with positive and negative ionization modes. The mobile phases consisted of H 2 O containing 0.05 % ammonia
(pH 11) in channel A, and 95 % acetonitrile containing 0.05 % ammonia in
channel B. Analysis were performed by running a linear gradient elution, starting
from 10 to 90 % B from 1 to 6.7 min, followed by a 1.3 min hold at 90 % B, a
10 % A, decreasing to 10 % B over 2 min, and holding for 3 min until the next
injection. The flow rate was 0.7 ml min
1 , and the injection volume was 10 l at a
column temperature of 40
ı C. All results are, as far as possible, evaluated without
distinction on instruments or methods.
Results and Discussion
Retention Times
PBI containing short aliphatic chains eluted too early in both ionization modes,
or did not ionize in negative mode at all. Two of the PBI’s; Hep-PBI and PentPBI, were tested further in the positive method, even though they differ chemically
from the lipophilic toxins. Hep-PBI coeluted with azaspirazid-1 (Aza-1) while PentPBI, which is slightly more polar than Hep-PBI, eluted between pectenotoxin-2
(PTX-2) and Aza-1. Because of this, it is reasonable to assume that Hep-PBI will be
appropriate as internal standard for Aza-1. The retention time of DHO was close to
dinophysistoxin-1 (DTX-1), which may indicate DHO as a suitable internal standard
for DTX-1.