Biology Reference
In-Depth Information
assay, the MCs consisted of [D-Asp
3
] MC-RR, [D-Asp
3
] MC-LR with Gly at position 1 instead of Ala
and combinations of homoarginine [hAr
2
] or [ADM Adda
5
] substitution (Wood
et al
., 2008).
The determination of CYN by HPLC has been described (Harada
et al
., 1994) with detection
at 262 nm but without the requirement of its extraction and concentration steps. Likewise, the
determination of CYN to 1 µg L
-1
with the help of LC/MS/MS did not require sample cleanup,
extraction and concentration steps (Eaglesham
et al
., 1999). Lyophilized cells of
Cylindrospermopsis
from Europe, Israel and Australia were analyzed for CYN content using HPLC. Extraction of CYN
was optimized with 5% formic acid to prevent interference in chromatograms by contaminating
compounds. Isocratic conditions of 5% (v/v) aqueous methanol plus 0.1% (v/v) trifl uoroacetic acid
as the mobile phase were used (Törökne
et al
., 2004).
ii) Mass spectrometric detection
:
During recent years, there has been a shift towards detection of
MCs by mass spectrometry (MS) following HPLC separation. The detection of MCs is unequivocal
because MCs produce characteristic ions in the mass spectra (Namikoshi
et al
., 1992a,b; Kondo
et
al
., 1992b; Rinehart
et al
., 1994; Lawton
et al
., 1995; Yuan
et al
., 1998, 1999a,b). MCs could be detected
by LC/MS/MS system at concentrations as low as 0.1 µg L
-1
that is ten times below the WHO
guidelines for MC-LR concentration in natural waters (Poon
et al
., 1993; Lawton
et al
., 1995; Kondo
et al
., 1992b, 1995; Tsuji
et al
., 1994 b; Bateman
et al
.,1995; Hormazabal
et al
., 2000; Ells
et al
., 2000;
Hummert
et al
., 2000; Zweigenbaum
et al
., 2000; Spoof
et al
., 2003; Dahlmann and Luckas, 2005).
By employing LC-MS, six MC variants have been identifi ed (Lawton
et al
., 1995). Tsuji
et al
.(1994b)
detected individual MCs to the level of 0.02 µg L
-1
by MS. Based on the mass spectrum of an available
standard, the identifi cation of a MC is possible. These data have to be additionally supplemented
by toxicity studies so as to enable the determination of toxicity equivalents. Unknown MCs could
also be identifi ed on the basis of fragmentation patterns by a variation of MS known as MS/MS
detection (Bateman
et al
., 1995; Lawton
et al
., 1995; Yuan
et al
., 1998, 1999a,b). Matrix-Assisted Laser
Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS) was used to identify
toxins (MCs, micropeptins and anabaenopeptolin) from intact microorganisms as well. Typing of the
blooms into toxic and non-toxic phenotypes can be achieved within few minutes (Erhard
et al
., 1997).
Besides providing information on molecular mass of all peptides, MALDI-TOF-MS also helped in
the detection of individual MC variants in cultures of
M
.
aeruginosa
(Robillot
et al
., 2000). Analysis
of intact colonies of
Microcystis
spp.
from bloom populations by MALDI-TOF-MS revealed the
identifi cation of a total of 46 individual peptides (21 of which have not been described previously).
In most colonies 2-10 individual peptides were detected. The principal component analysis revealed
MC-LR, MC-RR, MC-YR, anabaenopeptins B and E/F, a microviridin and a new cyanopeptolin.
Colonies of
M
.
aeruginosa
predominantly produced MCs whereas those of
M
.
ichthyoblable
were
negative for MCs but produced anabaenopeptins. No peptide metabolites could be detected in 16 of
the 19 colonies of
M
.
wesenbergii
(Welker
et al
., 2004b). Twenty six strains of
M
.
aeruginosa
collected
from potable water bodies of northern Portugal were isolated into pure cultures and the content
of MCs was determined using MALDI-TOF MS, ELISA and PCR procedure targeting genes of
mcy
gene cluster. Values of MCs ranged from 0.02 to 0.53% dry weight of cultures as revealed by ELISA.
MALDI-TOF-MS analysis demonstrated the presence of several chemically distinct variants of MCs
along with aeruginosins, anabaenopeptins and several other unidentifi ed peptide-like compounds
(Saker
et al
., 2005).
M
.
aeruginosa
B 2666, a strain that has not been investigated previously, released
into medium eight variants of MCs, i.e. MC-LR, MC-LA, [MeSer
7
] MC-LR, MC-LL, MC-LF, MC-
L(Aba), [Asp
3
] MC-LA and [Asp
3
] MC-LL. Of these, the last two MCs were reported for the fi rst
time separated by reversed-phase microbore liquid chromatography and introduced directly into a
