Biology Reference
In-Depth Information
hybrid linear ion-trap-Fourier transform ion cyclotron resonance mass spectrometer with electrospray
ionization. This technique has the following advantages. (1) It is fairly rapid. (2) It provides reliable
identifi cation in the absence of desired standards. (3) The work can be carried out even though one
does not possess additional information about structural variants from other analytical techniques
(Diehnelt
et al
., 2006).
Isolation and analysis of anatoxin-a and anatoxin-a(s) have been achieved by a four-step process
involving extraction with 0.05 M acetic acid followed by a reversed phase, ODS (octadecylsianized)
and a cation exchanger (COOH) organosilans bonded to silica gels were applied as a cleanup step.
Separation and identifi cation is achieved by TLC and HPLC chromatographies (Harada
et al
.,
1989). Thermospray-LC/MS was used to detect anatoxin-a that proved to be advantageous over
the conventional HPLC/UV. This method was found to be more suitable for detection of anatoxin-a
from cultures and naturally occurring bloom samples where the toxin could be detected at as low
concentrations as 500 pg (Harada
et al
., 1993). Further improvement in the detection of anatoxin-a
was made by the solid-phase extraction of the toxin that enabled a recovery of 75.7 ± 7.2% at a
concentration of 20 ng L
-1
. The detection of trace amounts of anatoxin-a from natural waters by
an automated on-line derivatization-LC-electrospray MS was reported where concentrations as
low as 2.1 ng L
-1
were detected (Takino
et al
., 1999). A new LC/MS method has been developed
for determination of anatoxin-a and homoanatoxin-a. The LC was coupled via an electron spray
ionisation (ESI) source to an ion-trap MS in positive mode. The detection limit for anatoxin was 0.03
ng (on-column) corresponding to 0.6 µg L
-1
(Furey
et al
., 2003a). Employing post-synaptic membrane
fractions of
Torpedo
electric tissue, the inhibition of binding of radioactively labelled bungarotoxin
by anatoxin-a and homoanatoxin-a (purifi ed from
Oscillatoria
sp. strain 193 PCC 9240 and
O
.
formosa
NIVA CYA-92 PCC 101111, respectively) to acetylcholine receptors was studied. This technique has
been termed as receptor radioligand-binding assay and has a high sensitivity with a dectetion limit
of about 1 x 10
-8
M which is equivalent to 1 µg L
-1
(≈6 x 10
-9
M) proposed for anatoxin-a in drinking
water. A concentration-dependent inhibition was observed with K
i
of 5.4 ± 1.1 x 10
-8
M for anatoxin-a
and 7.4 ± 0.9 x 10
-8
M for homoanatoxin-a (Aráoz
et al
., 2005). Anatoxin-a produced by
P
.
favosum
was identifi ed by HPLC with a UV-detector and by electrospray-Quadruple-Time of fl ight mass
spectrometry and further confi rmed by tandem mass spectrometry (Gugger
et al
., 2005).
Solid-phase microextraction of anatoxin-a using three forms of polyaniline fi lms (PANI) and a
single form of polypyrrole led to the identifi cation of leucoemeraldine form of PANI as best suited for
extraction. Optimization parameters for better extraction were 32 µm thickness, a salt concentration of
10% (w/v), an extraction time of 30 min and a stirring rate of 100 ppm (Ghassempour
et al
., 2005).
iii) Capillary Zone Electrophoresis (CZE)
:
Though capillary electrophoresis has been suggested for
the separation of MCs and nodularins (Boland
et al
., 1993; Onyewuenyi and Hawkins, 1996; Bouaicha
et al
., 1996; Bateman
et al
., 1995; John
et al
., 1997; Siren
et al
., 1999), it is not as sensitive as HPLC. Water
bloom samples and crude cyanobacterial extracts were subjected to CZE and micellar electrokinetic
chromatography (MEKC) that enabled simultaneous separation of anatoxin-a, MC-LR and CYN. The
detection limit of these toxins was in between 1-4 µg ml
-1
. Both CZE and MEKC were sequentially
applied for confi rming the results obtained by conventional techniques. Polyimide-coated fused-
silica capillary of 48.5 cm x 50 µm ID (40cm effective length) were used by hydrodynamic sample
introduction for injecting samples. The voltage applied was +25 kv at a temperature of 25ºC. The
measurements were made spectrophotometrically at 270 nm where diode array detection permitted
UV spectra to be recorded at 0.2 s intervals across the electropherogram. The separation of toxins
showed a dependence on the concentration of SDS in the running electrolyte. The application of
