158 / 302
2
In Korea, when an algal bloom is forecasted, samples
from the water supply source are collected and the
chlorophyll-a concentration and the cyanobacteria cell
number are measured. Based on the results, the situation
is categorized into one of the following situations: ‘algal
bloom watch,’ ‘algal bloom alert,’ or ‘algal bloom.’ In the
latter two situations, the cyanotoxins, mainly microcystin-LR,
are analyzed.
6
Accurate analysis of multiple samples
within a short time is required in order to monitor the
multiple points of the water supply source and each of
the processes taking place at water purification plants.
Traditionally, cyanotoxins have been measured by
performing extraction and concentration through
solid-phase extraction (SPE) followed by high-performance
liquid chromatography with ultraviolet detection (HPLC/
UVD) or photodiode array detection. More recently, the
analysis time has been reduced and the sensitivity improved
through the use of liquid chromatography–mass spectrometry
(LC-MS/MS) applying electrospray ionization (ESI).
7-13
The conventional SPE process required for all of these
methods uses a great deal of time and solvent.
An online preconcentration and injection method can
shorten the sample pretreatment process and help detect
trace amounts of target substances, while an Orbitrap-
type high-resolution mass spectrometry method takes into
account the retrospective aspect of data, making possible
both accurate identification of the analyzed toxins and
post-process quantitation of microcystin isomers.
Therefore, we combined these two techniques for the
identification and quantitation of microcystin-RR, -YR
and -LR as well as nodularin. Then, an optimized method
was developed to enhance the reliability and economic
efficiency by reducing the run time and the amount of
solvent necessary. The method was applied to raw and
treated water from water purification plants and river
systems.
Experimental
Reagents
Microcystin-LR, RR, and YR were procured from Wako
Pure Chemical Industries, Ltd. (Tokyo, Japan) in a dried
crystal form. Nodularin was procured from Cayman
Chemical (CA, USA) in a dissolved form (500 μg in 500 μL
of ethanol).
Information on each of the standard materials is summarized
in Table 1. Solvents were of residual pesticide grade.
Water was double distilled by reverse osmosis.
Standard Solutions and Calibration Curves
The standard solutions containing the cyanotoxins were
prepared by dissolving microcystin-LR, -RR, and -YR into
methanol at 100 μg/mL and by dissolving nodularin in
ethanol to a concentration of 10 μg/mL. Solutions were
stored in a cold room at 4 °C. Taking into consideration
the sensitivity of the analysis method and the WHO
guideline of a microcystin-LR concentration of 1 ng/mL,
the solutions were diluted into six different concentrations
within the range of 100 to 1000 pg/mL. An external
standard method was used for calibration curve verification
and sample identification. Then, the ratio of peak areas
according to the concentration of standard solution were
calculated.
Sample Collection and Storage
A total of 173 raw and treated water samples were collected
from 59 facilities at the Han (18 sites), Nakdong (18 sites),
and Geum-Seomjin (19 sites) Rivers, and in the city of
Geoje (4 sites), as well as 55 sites in the Han River basin
measurement network area. All samples were refrigerated
during transport, transferred directly to a cold room in
the lab, and maintained at 4 °C. Sample aliquots were
analyzed within three days of delivery.
Pretreatment and Instrumental Analysis
Online preconcentration using column switching was
applied as a means to minimize sample pre-treatment and
shorten analysis time. A
Thermo Scientific ™ EQuan MAX ™online sample concentration UHPLC-MS system equipped
with a Thermo Scientific ™ Hypersil GOLD aQ ™ preconcentration column (20 x 2.1 mm, 12 μm particle size) and a Thermo Scientific ™ Hypersil GOLD ™ analytical column (50 x 2.1 mm, 1.9 μm particle size) was used. Theallowable liquid sample injection range was 1 to 20 mL,
and in this study the sample injection amount was set at
1 mL after considering the WHO guideline, equipment
sensitivity, peak shape, and concentration ratio of the online
injection. The standard material for the calibration curve
and all the samples used in the analysis were filtered through
a 0.45 μm glass fiber (GF) membrane syringe filter.
A Thermo Scientific
™
Exactive
™
Orbitrap mass spectrometer
was operated in full-scan mode. Resolving power was set
to 50,000 (FWHM at
m/z
200). The detailed conditions
for the online sample concentration and injection and the
operation of the Orbitrap mass spectrometer are
summarized in Tables 2 and 3, respectively. For the
post-analysis identification and quantitation, an external
standard method was applied.
Compound
Name (CAS)
Formula
Molecular Weight
Microcystin
Microcystin-LR
(101043-37-2)
C
49
H
67
N
10
O
12
995.1717
Microcystin-RR
(111775-37-4)
C
49
H
75
N
13
O
12
1038.1997
Microcystin-YR
(101064-48-6)
C
52
H
72
N
10
O
13
1045.1873
Nodularin
Nodularin
(118399-22-7)
C
41
H
60
N
8
O
10
824.9627
Table 1. Chemical formula and molecular weight of target algal toxins