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A toxic cyanobacterial bloom usually consists of multiple
microcystin congeners in varying concentrations. Several
techniques for the analysis of microcystins have been
developed. Mouse bioassays, protein phosphatase
inhibition assays, and enzyme-linked immunosorbent
assays (ELISA) are effective for rapid screening but lack
specificity. Reversed-phase high-performance liquid
chromatography (HPLC) with ultraviolet (UV) detection
is the most common approach used for the separation,
detection and quantitation of microcystins. An ISO
method for microcystin analysis by HPLC-UV has been
validated for MC-RR, MC-YR and MC-LR.
4
However,
UV detection is susceptible to interferences from water
matrices and requires sample cleanup and concentration
to achieve desirable detection limits. Furthermore, UV-based
methods do not provide unequivocal identification of
known microcystins nor enable identification of unexpected
variants. Liquid chromatography in combination with
multi-stage mass spectrometry (LC-MS
n
) enables structural
characterization and unambiguous identification of trace
levels of microcystins. LC-MS/MS in multiple reaction
monitoring (MRM) acquisition mode allows highly selective
and sensitive quantitation and confirmation of target
microcystins, but this approach requires extensive
compound-dependent parameter optimization and cannot
be used to detect unexpected toxins. Full-scan MS/MS
approaches obviate the need for compound optimization
and enable determination of all microcystins present in a
sample.
The Thermo Scientific Velos Pro dual-pressure linear ion
trap mass spectrometer delivers sensitivity and speed for
qualitative and quantitative applications. High-quality
full-scan MS
n
spectra enable confident structural
elucidation and identification. Rapid scanning and fast
cycle times generate more scans across chromatographic
peaks for robust quantitation and allow the acquisition of
more MS
n
spectra in shorter chromatographic runs. A
wide dynamic range of up to six orders of magnitude
facilitates identification and quantitation of low-abundance
compounds in complex matrices. Complementary
fragmentation techniques may be performed in parallel to
enable more MS
n
information to be obtained from a single
sample. In this application note, we describe a simple and
sensitive targeted full-scan LC-MS/MS method for the
identification and quantitation of the microcystins MC-RR,
MC-YR, and MC-LR using the Velos Pro
™
ion trap mass
spectrometer coupled to a Thermo Scientific Dionex
UltiMate 3000 x2 Dual RSLC system.
Experimental
Sample Preparation
MC-RR, MC-YR and MC-LR standards were purchased
from Sigma-Aldrich®. A stock solution of a mixture of
these three microcystins was prepared at a concentration
of 5 µg/mL. Calibration solutions, with concentrations of
0.025 µg/L to 50 µg/L, were prepared by serial dilution
of the stock solution.
LC-MS/MS Analysis
A 50 µL sample was injected on a Thermo Scientific
Acclaim 120 guard cartridge with 150 L/min, washed for
two minutes to waste and then eluted onto a Thermo
Scientific PepMap100 analytical column for separation.
LC-MS/MS analysis was performed on an UltiMate
™
3000 x2 Dual RSLC system coupled to an Velos Pro mass
spectrometer.
LC Parameters
Guard cartridge:
Acclaim
™
120 C18 (10 x 3.0 mm i.d., 5.0 µm
particle size, 120 Å pore size)
Analytical column:
Acclaim PepMap100 C18 (150 x 1.0 mm i.d.,
3.0 µm particle size, 100 Å pore size)
Mobile Phase A:
Water containing 0.1% formic acid
Mobile Phase B:
Acetonitrile containing 0.1% formic acid
Column temperature:
40 °C
Sample injection volume: 50 µL
Flow rate:
150 µL/min
Gradient:
Table 1
Table 1: LC Gradient
MS Parameters
Ionization mode:
Positive electrospray ionization (ESI)
Collision energy:
35%
Isolation window:
2
Targeted full-scan MC-RR [M+2H]
2+
at
m/z
520 [
m/z
150-1100]
MS/MS:
MC-YR [M+H]
+
at
m/z
1045 [
m/z
285-1100]
MC-LR [M+H]
+
at
m/z
995 [
m/z
285-1100]
Time
% A
% B
0.1
98
2
1.5
98
2
2.0
80
20
3.0
60
40
7.4
40
60
7.5
2
98
7.9
2
98