O
R
N
H
NH
O
OH
CH3
HN
CH3
H
N
CH3
Microcystin-LR
CH3
OCH3
CH3
NH
N
O
CH3
HN
CH2
NH
N
H
NH2
OH
CH3
R = C
H
CH3
NH
Microcystin-RR
R = H 2N
OH
Microcystin-YR
N
H
CH2
R=
27806
EQUIPMENT
Dionex UltiMate 3000 HPLC system including:
DGP-3600A pump with SRD 3600 solvent rack
with degasser
WPS-3000TSL semiprep autosampler
(with 2500 L sample loop)*
TCC-3200 Thermostatted Column Compartment
equipped with two 2p6p valves
VWD-3400RS UV-vis detector
Chromeleon software
Orion 420A+ pH meter, Thermo Scientific
*The analytical version of the WPS-3000TSL Autosampler can also
be converted and used for large-volume injection for on-line SPE. The
procedure is the same as specified in Reference 6.
REAGENTS
Deionized water, Milli-Q Gradient A10,
Millipore Corporation
Acetonitrile (CH3CN) and methanol (CH3OH),
HPLC grade, Fisher
Potassium dihydrogen phosphate (KH2PO4), dipotassium
hydrogen phosphate (K2HPO4), and phosphoric acid
(H3PO4), 85% (analytical grade), SCRC, China
STANDARDS
100 g of microcystins-LR (CAS 101043-37-2),
-RR (CAS 111755-37-4), and -YR (CAS 101064-48-6),
respectively, 95% (HPLC), Alexis Corporation
Prepare stock standard solutions with 50 g/mL
concentrations by dissolving the standards with 2000 L
of methanol. Prepare the standard solutions used for the
calibration curve by making appropriate dilutions of the
stock standard solutions with water.
SAMPLES
Tap water samples were collected at the Dionex
Shanghai Applications Lab. The lake water sample
was collected at Zhangjiang High-Science and
Technology Park located in the Pudong District of
Shanghai, China. Bottled spring water samples were
purchased from a supermarket in Shanghai. These
samples were filtered through a 0.45 m membrane
(Millex-HN) prior to injection.
CHROMATOGRAPHIC CONDITIONS
On-Line SPE
Column: Acclaim PA2, 3 m, 3.0 33 mm
(P/N 066276)
Analytical
Column: Acclaim PA2, 3 m, 3.0 150 mm
(P/N 063705)
For SPE:
A: 22.5 mM KH2PO42.5 mM K2HPO4
buffer (dissolve ~ 3.1 g of KH2PO4 and
0.44 g of K2HPO4 in 1 L of water)
For separation:
A: 0.05% (v/v) H3PO4 (dilute 0.6 mL
of 85% H3PO4 to 1 L with water)
B: CH3CN
In gradient (Table 1)
Valve-Switching: Table 1
Column Temp.: 40 C
Mobile Phase:
Flow Rate:
Injection Vol.:
UV Detection:
Absorbance at 240 nm
B: CH3CN
In gradient (Table 1)
Table 1. Gradients and Valve Switching for Target-Cut On-Line SPE and Separation
Time
(min)
Solvent A
Buffer
(%)
Solvent B
CH3CN
(%)
Solvent A
Buffer
(%)
Valve Switching
Solvent B
CH3CN
(%)
Left
Right
0.00
85
15
80
20
1-2
5.00
80
20
6.95
6-1
7.00
85
15
65
35
7.35
1-2
7.50
0.7
0.7
6-1
20
80
8.50
20
80
8.60
80
20
12.0
41
59
12.1
85
15
15.0
85
15
80
20
Table 2. Gradient and Valve Switching for Traditional On-Line SPE and Separation
Time (min)
Solvent A
Buffer
(%)
Solvent B
CH3CN
(%)
Valve
Switching
Solvent A
Buffer
(%)
Solvent B
CH3CN
(%)
0.00
80
20
1-2
5.00
80
20
6-1
6.00
1-2
9.00
50
50
25
75
11.0
25
75
11.1
80
20
12.0
80
20
9.10
0.7
0.7
80
20
27807
The Acclaim Polar Advantage II (PA2) is a polarembedded column designed for enhanced hydrolytic
stability within a wide range of pH values (pH 1.5 to 10),
and compatibility with 100% aqueous mobile phases,
overcoming the limitations of conventional C8 and C18
reversed-phase columns.
Effect of Buffer pH Value
The pH value of the mobile phase buffer may affect
the retention of microcystins-RR, -YR, and -LR. Changes
in their retention behavior on the Acclaim PA2 stationary
phase were investigated. Experiments showed that when
the buffer pH value decreased from pH 6.5 to 2.7, the
retention time of microcystins-YR and -LR increased
and the resolution between them improved, whereas the
retention time of microcystin-RR did not change. The
three microcystins were separated at a pH value lower
than 2.5. They co-eluted at approximately pH 6.0.
Thus, for the requirements addressed here, the
PA2 column is a good choice as an SPE column for
concentrating the three microcystins from large-volume
water samples (tap water and beverages) and co-eluting
them using mobile phase buffer with a high pH value
(~ 6.0). The PA2 column is also a good choice as an
analytical column for the separation using a mobile phase
buffer with a low pH value.
Microcystin-RR
Flow Rates:
Injection Vol.:
Detection:
20
mAU
Microcystin-YR
Microcystin-LR
SPE Column:
Anal. Column:
Column Temp.:
Eluent for SPE:
0 b
10
6.5
Microcystin-RR
10
mAU
Position of
Right Valve
10
Minutes
Description
1-2
1-2
6-1
1-2
6-1
6.5
Position of
Left Valve
12
a
-6
6-1
Microcystin-YR
Microcystin-LR
-6
27809
12
13
27808
Figure 3. Chromatograms of a) bottled spring water, b) tap water, and c) lake water spiked with 1 g/L each of microcystin-RR,
-YR, and -LR standard using A) traditional and B) target-cut
on-line SPE methods.
Microcystin-RR
Flow Rates:
Injection Vol.:
Detection:
30 A
mAU
Microcystin-YR
Microcystin-LR
SPE Column:
Anal. Column:
Column Temp.:
Eluent for SPE:
0 1
-20
15
10
6.5
20
mAU
0
-2
Microcystin-YR
Microcystin-LR
Microcystin-RR
8 B
6.5
12
10
Minutes
14
15
27810
mAU
10
10
11
10
11
Microcystin-RR
50 B
mAU
7.00 min
tvalve-switching 1
-10
6.5
6.8
0.45 min
Wh
7.2
Minutes
7.45 min
tvalve-switching 2
7.4
50 C
7.6
7.8
8
27811
mAU
3
-5
6.5
12.5
12.5
Microcystin-YR
2
-5
6.5
11
Microcystin-YR
1
-5
6.5
mAU
Microcystin-LR
Microcystin-RR
50 A
Microcystin-LR
Flow Rates:
Injection Vol.:
Detection:
Microcystin-YR
Minutes
Microcystin-LR
SPE Column:
Anal. Column:
Column Temp.:
Eluent for SPE:
50
12.5
27812
Microcystin-RR
6 A
mAU
Flow Rates:
Injection Vol.:
Detection:
10
mAU
0
-1
MC-YR
MC-LR
Flow Rates:
Injection Vol.:
Detection:
MC-RR
SPE Column:
Anal. Column:
Column Temp.:
Eluent for SPE:
Microcystin-LR
Anal. Column:
Column Temp.:
Eluent for SPE:
SPE Column:
1
3
6
4
5
2
6.5
12
10
14
Minutes
0
-20
7.5
10
11
12
13
14
15
15
27814
Figure 9. Overlay of chromatograms of six consecutive injections of a drinking water sample spiked with 0.5 g/L each of
microcystin-RR, -YR, and -LR standard using the target-cut
on-line SPE method in Table 1.
mAU
0
-2
Microcystin-YR
Microcystin-LR
Microcystin-RR
8 B
Microcystins
5.5
9
Minutes
10
11
14
13
27813
Retention
Time RSD
Peak Area
RSD
Concentration
of standard
(g/L)
RR
0.037
1.53
0.5
YR
0.028
1.59
0.5
LR
0.029
1.13
0.5
Microcystin-RR
mAU
-0.5
10
Microcystin
Microcystin-YR
Microcystin-LR
SPE Column:
Anal. Column:
Column Temp.:
Eluent for SPE:
11
Regression
Equations
r (%)
RR
A = 0.0844
c - 0.0027
99.997
YR
A = 0.1054
c - 0.0022
99.994
LR
A = 0.0942
c + 0.0030
99.994
MDL*
RSD for
Range of
Standards Calibration (g/L)
Curve
g/L
0.110
0.91
0.028
1.25
0.028
1.21
0.019
Note. * The single-sided Students test method (at the 99% confidence limit)
was used for determining MDL, where the standard deviation (SD) of the
peak area of six injections is multiplied by 4.03 to yield the MDL.
12
12.2
12.5
27815
Figure 10. Chromatogram of a mixed solution with concentrations of 0.1 g/L each of microcystin-RR, -YR, and -LR standard
using the target-cut on-line SPE method in Table 1.
Microcystin-RR
8 A
mAU
10
10
10
12
Microcystin-RR
8 B
mAU
b
-1 a
6.5
8
12
Microcystin-RR
mAU
b
a
-1
6.5
CONCLUSION
This work describes a target-cut on-line SPE method
that can fully recover low concentrations (< 1 g/L) of
three microcystins (-RR, -YR, and -LR) when added to
three different water samples. These concentrations are
less than the maximum concentrations recommended
by WHO. This method is fully automated and easily
configured on an UltiMate 3000 2 Dual HPLC system.
14
15
14
15
14
15
Microcystin-YR
Microcystin-YR
Microcystin-YR
Microcystin-LR
b
a
-1
6.5
Sample Analysis
SPE Column:
Anal. Column:
Column Temp.:
Eluent for SPE:
12
Minutes
27816
Tap Water
Detected
(g/L)
Added
(g/L)
Found
(g/L)
Lake Water
Recovery
(%)
Detected
(g/L)
Added
(g/L)
Found
(g/L)
Detected
(g/L)
Found
(g/L)
Recovery
(%)
RR
ND
0.50
0.48
96
ND
0.50
0.55
110
ND
0.50
0.49
98
YR
ND
0.50
0.46
92
ND
0.50
0.51
102
ND
0.50
0.48
96
LR
ND
0.50
0.48
96
ND
0.50
0.51
102
ND
0.50
0.49
98
10
Added
(g/L)
REFERENCES
1. Cyanobacterial Toxins: Microcystin-LR in
Drinking-Water; Background Document for
Development of WHO Guidelines for DrinkingWater Quality: World Health Organization, 2003.
2. Lee, H.S.; Jeong, C.K.; Lee, H.M.;Choi, S.J.;
Do, K.S.; Kim, K.; Kim, Y.H. On-Line Trace
Enrichment for the Simultaneous Determination
of Microcystins in Aqueous Samples Using HighPerformance Liquid Chromatography with DiodeArray Detection. J. Chromatogr. A 1999, 848, 179.
3. Aguete, E.C.; Gago-Martnez, A.; Leo, J.M.;
Rodrguez-Vzquez, J.A.; Menrd, C.; Lawrence, J.F.
HPLC and HPCE Analysis of Microcystins RR, LR,
and YR Present in Cyanobacteria and Water by Using
Immunoaffinity Extraction. Talanta, 2003, 59, 697.
4. Kondo, F.; Matsumoto, H.; Yamada, S.; Tsuji, K.;
Ueno, Y.; Harada, K. Immunoaffinity Purification
Method for Detection and Quantification of
Microcystins in Lake Water. Toxicon. 2000, 38, 813.
5. Tsutsumi, T.; Nagata, S.; Hasegawa, A.; Ueno,
Y. Immunoaffinity Column as Clean-Up Tool for
Determination of Trace Amounts of Microcystins in
Tap Water. Food Chem. Toxicol. 2000, 38, 593.
6. Dionex Corporation, Determination of Vitamin
B12 in Beverages Using On-Line SPE Followed by
HPLC with UV Detection. Application Note 256,
LPN 2574, 2010, Sunnyvale, CA.
7. Dionex Corporation, Determination of Water- and
Fat-Soluble Vitamins by HPLC. Technical Note 89,
LPN 2598, 2010, Sunnyvale, CA.
8. Sun, Q.L; Zhuang, S.Q. Determination of Vitamin
B12 Using UPLC; Application Summary for Waters
Corporation: Milford, MA, 2010.
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