200
T. Jauffrais et al.
AZAs Harvesting Procedures
Tangential flow filtration (Sartorius Stedim Biotech, Sortojet Pump with Sartocon
Slice and 5 0.1 m
2 Hydrosart Open Channel Microfiltration Cassettes) was applied
to separate the algae from the culture medium. Thus, 200 L of algal culture were
divided into 1 L of algal concentrate (retentate) and almost 200 L of permeate
(Fig. 17.1).
For toxin extraction from the retentate, the algal concentrate was sonicated
(20 min in ice, Bioblock Scientific, Vibra-cell 75115), 25 g of activated Diaion HP20
polymeric resin was added, and gently agitated within the algal concentrate over
24 h, on a laboratory shaker (IKALABORTECHNIK, KS125basic). The resin was
then washed with 1 L of Milli-Q water (Millipore, Integral 3 system), and placed in
a glass column (3 by 60 cm). The toxin was eluted with three volumes of acetone
(50 mL) at 1 mL min
1 . The extract was then evaporated using a rota-evaporator
(B¨ uchi, Rotavapor R-200) and the residue was reconstituted in 5 mL methanol.
For toxin extraction from the 200 L permeate, two procedures were tested:
1. Passive samplers were placed into the permeate as developed by MacKenzie et al.
(2004) and Fux et al. (2008, 2009). Eight SPATT bags (solid phase adsorption
toxin tracking) containing 3 g of activated Diaion
® HP20 resin were added into
the permeate and gently agitated within a submerged pump over 72 h. The resin
was then extracted as above.
2. A submerged pump (20 L min
1 ) was placed into the permeate and connected to
a column containing 25 g of activated Diaion
® HP20 resin over 72 h. The resin
was then extracted as above. This procedure was an adaptation of Rundberget
et al. (2007) developed for large scale extraction of micro-algal biotoxin in situ.
LC-MS/MS Analysis
The samples were analyzed by LC/MS-MS using an Agilent 1100 model coupled
to a triple quadrupole mass spectrometer (SCIEX-Applied Biosystems, API 2000)
for quantification of AZAs. Five microlitre of each sample were injected into
the LC-MS/MS, toxins were separated by reversed-phase chromatography with a
silica-based column (Hypersil BDS C8 column, size 50*2 mm, 3 m particle size;
Phenomenex) The A and B mobile phases were 100 % water and acetonitrile/water
(95/5, v/v) respectively, both containing 2 mM ammonium formate and 50 mM
formic acid. The BDS-Hypersil column was eluted isocratically at a dilution rate of
250 L min
1 (75%B) at 20
ı C for 10 min.
AZAs were quantified by comparison with a series of AZA-1 standard from the
National Research Council, Canada (NRC). The two most intense product ions were
selected with the following transitions: AZA1 m/z 842.5 > 824.5 and 842.5 > 672.4,
and AZA2 856.5 > 838.5 and 856.5 > 672.4
T. Jauffrais et al.
AZAs Harvesting Procedures
Tangential flow filtration (Sartorius Stedim Biotech, Sortojet Pump with Sartocon
Slice and 5 0.1 m
2 Hydrosart Open Channel Microfiltration Cassettes) was applied
to separate the algae from the culture medium. Thus, 200 L of algal culture were
divided into 1 L of algal concentrate (retentate) and almost 200 L of permeate
(Fig. 17.1).
For toxin extraction from the retentate, the algal concentrate was sonicated
(20 min in ice, Bioblock Scientific, Vibra-cell 75115), 25 g of activated Diaion HP20
polymeric resin was added, and gently agitated within the algal concentrate over
24 h, on a laboratory shaker (IKALABORTECHNIK, KS125basic). The resin was
then washed with 1 L of Milli-Q water (Millipore, Integral 3 system), and placed in
a glass column (3 by 60 cm). The toxin was eluted with three volumes of acetone
(50 mL) at 1 mL min
1 . The extract was then evaporated using a rota-evaporator
(B¨ uchi, Rotavapor R-200) and the residue was reconstituted in 5 mL methanol.
For toxin extraction from the 200 L permeate, two procedures were tested:
1. Passive samplers were placed into the permeate as developed by MacKenzie et al.
(2004) and Fux et al. (2008, 2009). Eight SPATT bags (solid phase adsorption
toxin tracking) containing 3 g of activated Diaion
® HP20 resin were added into
the permeate and gently agitated within a submerged pump over 72 h. The resin
was then extracted as above.
2. A submerged pump (20 L min
1 ) was placed into the permeate and connected to
a column containing 25 g of activated Diaion
® HP20 resin over 72 h. The resin
was then extracted as above. This procedure was an adaptation of Rundberget
et al. (2007) developed for large scale extraction of micro-algal biotoxin in situ.
LC-MS/MS Analysis
The samples were analyzed by LC/MS-MS using an Agilent 1100 model coupled
to a triple quadrupole mass spectrometer (SCIEX-Applied Biosystems, API 2000)
for quantification of AZAs. Five microlitre of each sample were injected into
the LC-MS/MS, toxins were separated by reversed-phase chromatography with a
silica-based column (Hypersil BDS C8 column, size 50*2 mm, 3 m particle size;
Phenomenex) The A and B mobile phases were 100 % water and acetonitrile/water
(95/5, v/v) respectively, both containing 2 mM ammonium formate and 50 mM
formic acid. The BDS-Hypersil column was eluted isocratically at a dilution rate of
250 L min
1 (75%B) at 20
ı C for 10 min.
AZAs were quantified by comparison with a series of AZA-1 standard from the
National Research Council, Canada (NRC). The two most intense product ions were
selected with the following transitions: AZA1 m/z 842.5 > 824.5 and 842.5 > 672.4,
and AZA2 856.5 > 838.5 and 856.5 > 672.4