Subscriber access provided by - Access paid by the | UCSB Libraries
Article
Rapid Microcystins Determination Using a Paper Spray Ionization Method with Time-of-Flight Mass Spectrometry System Xiaoqiang Zhu, Zhengxu Huang, Wei Gao, Xue Li, Lei Li, Hui Zhu, Ting Mo, Bao Huang, and Zhen Zhou J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b02101 • Publication Date (Web): 25 Jun 2016 Downloaded from http://pubs.acs.org on June 28, 2016
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 26
Journal of Agricultural and Food Chemistry
1
Rapid Microcystins Determination Using a Paper Spray Ionization
2
Method with Time-of-Flight Mass Spectrometry System
3 4
†,‡ *, †, § †, § †, § †, § # Xiaoqiang Zhu , Zhengxu Huang , Wei Gao , Xue Li , Lei Li , Hui Zhu ,
5
# # †, § Ting Mo , Bao Huang , Zhen Zhou
6 7 8 9 10 11 12
†
Institute of Atmospheric Environment Safety and Pollution Control, Jinan University,
Guangzhou 510632, China ‡ §
Department of Ecology, Jinan University, Guangzhou 510632, China Guangdong
Provincial
Engineering
Research
Center
for
On-line
Source
Apportionment System of Air Pollution, Guangzhou 510632, China #
Guangzhou Hexin Analytical Instrument Co.,Ltd,Guangzhou 510530, China
13
*Corresponding author (Tel: +8602082071910; Fax: +8602082071902; E-mail:
14
[email protected])
15 16 17 18 19 20 21 22 1
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 2 of 26
23
ABSTRACT: The eutrophication of surface water sources and climate changes have
24
resulted in an annual explosion of cyanobacterial blooms in many irrigating and
25
drinking water resources. To decrease health risks to the public, a rapid real time
26
method for the synchronously determination of two usual harmful microcystins
27
(MC-RR and MC-LR) in environmental water samples was built by employing a paper
28
spray ionization method coupled with a time-of-flight mass spectrometer system.
29
With this approach, direct analysis of microcystins mixtures without sample
30
preparation has been achieved. Rapid detection was performed simulating the
31
release process of microcystins in reservoirs water samples, and the routine
32
detection frequency was every 3 minutes. The identification time of microcystins was
33
reduced from several hours to few minutes. The limit of detection is 1 μg/L and the
34
limit of quantitation is 3 μg/L. This method displays the ability for carrying out rapid,
35
direct and high-throughput experiments for determination of microcystins, and it
36
would be of significant interest for environmental and food safety applications.
37
Microcystins;
38
KEYWORDS:
39
Simultaneous analysis;
Paper
spray
ionization;
40 41 42 43 44 2
ACS Paragon Plus Environment
Rapid
determination;
Page 3 of 26
Journal of Agricultural and Food Chemistry
45 46
■INTRODUCTION For the past few years, with the industrial and agricultural development, water 1,2
47
eutrophication is becoming more and more serious in China.
48
eutrophication of surface water sources and climate changes have result in
49
cyanobacterial blooms in many drinking and irrigating water resources.
50
The aggravated
Microcystins are cyanotoxins with cyclic heptapeptide structures. The common
51
structure
is
cyclo(D-Ala-X-D-MeAsp-Z-Adda-D-Glu-Mdha)
in
which
Adda
is
52
(2S,3S,8S,9S)-3-amino-9-methoxy-2,6,8-trimethyl-10-phenyl-deca-4, 6-dienoic acid,
53
Mdha is N-methyldehydroalanine, and D-MeAsp is D-erythromethylaspartic acid.
54
Studies on the structure and identification of microcystins have been evolving from
55
the early 1960s, identifying more than 90 variants of microcystins. Microcystin-LR
56
(MC-LR) and microcystin-RR (MC-RR) are among the most common and harmful
57
forms of cyanobacterial blooms in aquatic environments (Figure 1).6 Cropland can be
58
polluted by irrigating with microcystin-contaminated water. These toxins can be
59
accumulated in different organisms and crops and can easily reach human consumers.
60
Previous studies have shown that the consumption of aquatic products, drinking
61
water and crops are important exposure routes of microcystins to humans.6
62
Microcystins have the function of liver toxicity and tumor promotion. They not only have a
63
toxic effect on aquatic organisms, but also harmful to human health through drinking water
64
and food chain. Microcystins are particularly prevalent in eutrophic waters following
65
bloom lysis, with a wide range of concentrations (19−141 μg/L) in various water
66
bodies throughout the world.
3-5
7
7,8
More than 60,000 human intoxications due to 3
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
67
Page 4 of 26
aquatic toxins occur per year, with a 1.5% mortality rate.9
68
Microcystins have been continually studied mainly due to their toxicity to
69
human and enhanced occurrence frequency. The monitoring of the quality of water
70
bodies to assess the presence of microcystins is of the utmost importance and often
71
difficult because cyanobacterial blooms may generate many different kinds of
72
microcystins. 10 For successful treatment of microcystins and to minimize the risk to
73
human health, sensitive and reliable methods able to detect the different types of
74
microcystins are urgently required. Several methods for microcystins determination,
75
such as biological-based screening methods and analytical techniques, have been
76
developed.11-14 However, these methods may be improved by developing more rapid
77
and efficient approaches.
78
There are two main approaches to detect the presence of microcystins:
79
bio-chemical assays and instrument analytical methods. Biochemical assays such as
80
invertebrate
81
measurements, can detect the total amount of microcystins in samples. However,
82
they are incapable of distinguishing microcystin analogues and may be strongly
83
affected by matrix effects.
84
performance liquid chromatography (HPLC)12 and HPLC coupled to mass
85
spectrometry (HPLC-MS),13 are effective and powerful technologies to detect
86
microcystins in complex matrices. 14-16 The development of electrospray ionization
87
(ESI) source combined to LC has provided an easy mass detection method to ionize
88
non-volatile materials. However, methods based only on HPLC alone fail to provide
bioassay,
protein
11
phosphatase
inhibition
assays,
and
ELISA
Instrumental analytical methods, such as high
4
ACS Paragon Plus Environment
Page 5 of 26
Journal of Agricultural and Food Chemistry
89
structural information. Moreover, many of the classical analytical methods usually
90
need complex sample pretreatment in order to remove the reagents used and
91
derivatization for HPLC analysis. 17,18
92
Ambient ionization methods, such as extractive electrospray ionization (EESI),
93
desorption electrospray ionization (DESI), and others, have been used for rapid
94
analysis of complex analyte without sample preparation.19-21 The paper spray
95
technique is an ambient ionization method in which sample storage, separation and
96
ionization processes are completed on a piece of paper with a sharp point.22
97
We decided to perform this study to establish a rapid method for the detection
98
of microcystins in water. The identification of microcystins has been developed using
99
a time-of-flight mass spectrometer system with paper spray ionization methods, and
100
the identification time was reduced to a few minutes compared with HPLC/MS-based
101
methods. Two microcystins, MC-LR and MC-RR, were used for method development
102
and validation. This method represents a powerful tool to perform rapid, real time
103
and high-throughput assays for selective screening of microcystins.
104
■MATERIALS AND METHODS
105
Instrumentation and Chemicals. All chemicals used in this study were analytical
106
grade reagents. MC-RR and MC-LR were purchased from Taiwan Algal Science
107
(Taoyuan, China) and dissolved in methanol. Chromatography paper(Grade 1) was
108
bought from Whatman(Guangzhou, China).
109
All experiments were performed with a homemade time-of-flight mass
110
spectrometer (resolution>10,000 FWHM,FWHM means full width at half maximum, 5
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
111
mass accuracy of 2 ppm,with in-source collision-induced dissociation function).
112
Spectra were collected setting the instrument in positive mode.
113
Environmental water samples
114
Environmental water samples were collected from Helong reservoir and Tianlu Lake
115
in Guangzhou, China (Figure 2). Sampling time was around May and June, 2016, the
116
main season for cyanobacterial blooms. The top 15 cm of the water column was
117
sampled, and stored in non-transparent glass bottles.
118 119 120 121
A simple sample pretreatment was performed to determine total microcystins following these two steps: Step 1: Environmental water samples were entirely mixed to ensure that algae
cells and sediments were evenly distributed.
122
Step 2: 500 mL of samples were transferred to a 1000 mL round flask. Samples
123
were frozen and thawed 3 times by holding the flask alternately in a dry ice-acetone
124
bath followed by a warm water bath (30 °C).
125
Paper spray ionization and mass spectrometry. Paper spray ionization was used as
126
reported in literature. 23, 24 Chromatographic paper was chosen as substrate to apply
127
the sample. Paper was cut into a equilateral triangle (height 11mm, base 10mm).
128
The space between the inlet of the mass spectrometer and the tip of the paper
129
was 5 mm. The high voltage was applied by a copper clip. The sample was loaded
130
onto the paper by using a dropper. Loaded sample was then air-dried, methanol was
131
added and a voltage of 3.5 kV (DC) was applied via the copper clip. Sample was thus
132
ionized and sprayed into the mass spectrometer. Measurements were performed in 6
ACS Paragon Plus Environment
Page 6 of 26
Page 7 of 26
Journal of Agricultural and Food Chemistry
133
the range m/z 100-1500. The instrument was externally calibrated using PEG 1000>.
134
The fragmentation was performed with the in-source collision-induced dissociation
135
function by using the following parameters: focus voltage, 132V; start voltage, 100V;
136
end voltage, 70V; out-plate voltage, 93V; skimmer voltage, 35V.
137
Rapid and Simultaneous determination of microcystins. The release process
138
mimicking the release process of the microcystins in the environment was simulated
139
in laboratory following these three steps:
140 141 142 143
Step 1: 500 mL environmental water sample were spiked with 100 μg/L of standard MC-RR and 50 μg/L of standard MC-LR and poured into a beaker; Step 2: Environmental water was continuously added into a beaker at a rate of 30 mL/min;
144
Step 3: Samples (5 parallel samples for each) were obtained at regular intervals
145
(3 min) from the beaker. These samples were analyzed for the presence of
146
microcystins. Detection time for one sample is only 1 min, therefore the detection
147
frequency can be adjusted as required.
148
■RESULTS AND DISCUSSION
149
Method Development. The primary challenge in method development was to find a
150
suitable set of conditions that would allow all microcystins to be analyzed
151
simultaneously from a single sample. The method was optimized in terms of spray
152
voltage, paper size and apex angle.
153
Spray voltage should be as low as possible, under the premise of producing
154
stable spray and ion current. Lower voltages are also desirable for lowering the 7
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 8 of 26
155
background produced by chemical noise. Paper spray became completely unstable
156
below 2.5 kV, thus 3.5 kV was used.
157
Based on previous studies, the paper size and apex angle were investigated.
25
158
The paper size has little effect on signal strength and stability, but it changes the
159
amount of solvent that can be absorbed. Compared with paper size, the apex angle is
160
a more important parameter, affecting the spectrum quality in a large extent. The
161
apex angle influences the emission of charged droplets and the strength of the
162
electric field as well as the competitive process of gaseous discharge. A more acute
163
angle resulted in greater currents suggesting that the electric field strength exceeded
164
the threshold required for stable electrospray and resulted in a gaseous discharge.
165
According to experience, this kind of discharge will increase the complexity and noise,
166
which has a negative effect on the quality of the spectrum. Therefore, a less acute
167
angle of 50° was the better choice under the conditions tested.
168
Method validation. The matrix effect (ME) is affected by many factors, such as
169
the type of matrix, the treatment method and the instrument.6 The ME was
170
evaluated by contrasting the calibration curves of microcystin standards in different
171
solvent. The signal affect for microcystins was calculated using following equation:
172
S −S ME = 100 × m s % SS
Eqn. 1
173
The Sm represents the slopes of the matrix-matched calibration curves. Ss is the
174
slopes of the solvent-only situation. The matrix effect can be ignored when ranging
175
from −10% to +10%, it is considered minor when ranging from −20% to −10% or from
176
+10% to +20%, medium when ranging from −50% to −20% or from +20% to +50%, 8
ACS Paragon Plus Environment
Page 9 of 26
Journal of Agricultural and Food Chemistry
177
and intense when it is more than ±50%.6 In our case, the impacts of matrix on the
178
microcystins were −9% to +12%.
179
MC-RR and MC-LR standard solutions were used to assess the linearity and
180
range of measurement 25. Seven standard solution concentrations (10, 20, 30, 40, 60,
181
80, 100 μg/L) were analyzed to estimate the linearity and each point was detected
182
three times. The regression lines of peak areas on different concentrations were
183
obtained by least squares method (Figure 3). Estimated limits of detection (LODs)
184
ranged from 1 to 5 μg/L. The LODs of MC-RR and MC-LR were confirmed by detecting
185
spiked samples at the minimum concentration of 1 μg/L, with an S/N ratio >3. In the
186
same way, the limits of quantitation (LOQs) are defined as 3 μg/L (S/N >10).
187
Characterization of MC-LR and MC-RR. The molecular ion at m/z 996.2 [M+H]+ was
188
observed in the mass spectrum of MC-LR (Figure 4), for the existing of arginine
189
residue, in which the guanidine group decides the ionization state of target
190
analytes.26 The double-charged molecular ion at m/z 520.1 [M+2H]2+ was observed in
191
the spectrum of MC-RR, which contains two basic arginine residues (Figure 4).27
192
The fragmentation of precursor ions of MC-LR and MC-RR gave product ions at
193
m/z 135.2 and 213.3, respectively. The fragment ion at m/z 135.2 was recognized as
194
the [phenyl-CH2−CH(OCH3)]+, formed by the a-cleavage at the methoxy group of the
195
Adda β-amino acid moiety.28 The product ion at m/z 213.3 was recognized as
196
[Glu-Mdha+H]+ produced by by cleavage of the N-methyldehydroalanine and α-linked
197
glutamic acid. These findings confirm previously reported data. 25, 29
198
Detection of microcystins in environmental water samples. Sufficient microcystins 9
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
199
recovery cannot be Achieved without a cell lysis procedure, for microcystins are
200
commonly membrane-bound or intracellular toxins in algae cells. For example, in log
201
phase populations more than 90% of microcystins are intracellular. Total sample
202
microcystins concentrations is an important index to quality guidelines for drinking
203
water. Microcystins can be released into water if algal cells are destroyed.
204
Environmental water samples, subjected to a cell lysis procedure, unspiked, were
205
determined by the paper spray ionization method. In the positive mode, molecular
206
ion at m/z 996.2 [M+H]+ was typical for MC-LR, while the double charge at m/z 520.1
207
[M+2H]2+ was typical of MC-RR, showing a splitting in the isotope pattern of 0.5 m/z
208
(Figure 5). If microcystins are present in a complex matrix, their detection is more
209
difficult. However, paper spray ionization coupled to mass spectrometry. Is a robust
210
approach based on molecular and fragment ions typical of microcystins that can be
211
quickly detected. These results are consistent with our previous experiments and
212
with other studies.19,23 Paper spray ionization is a particularly simple ambient
213
ionization technique which can be employed to measure trace constituents of
214
complex mixtures. In the analysis performed on the five sampling sites, the
215
predominant toxin detected was MC-RR, with a range of concentration of 0-9.1 μg/L.
216
MC-LR was detected in ranges from 0-5.8 μg/L (Table 1).
217
Rapid and Simultaneous detection of microcystins in a complex mixture. The
218
monitoring of the quality of water bodies to assess the presence of cyanotoxins is
219
often difficult considering that cyanobacterial blooms may contain complex mixtures
220
of microcystins. 10
ACS Paragon Plus Environment
Page 10 of 26
Page 11 of 26
Journal of Agricultural and Food Chemistry
221
The Microcystins release process was simulated in the laboratory by spiking
222
standard MC-LR and MC-RR in water samples. Microcystins concentration was
223
determined with our method based on paper spray ionization and mass
224
spectrometry and calculated using standard curves for samples collected every 3
225
minutes. Using five parallel samples, standard deviation and standard error were
226
calculated. In Figure 6 are reported measured concentrations for MC-RR and MC-LR.
227
Both of the microcystins concentrations show a declining trend, with MC-RR
228
concentration double that of MC-LR. Although the routine detection frequency is
229
every 3 min, one whole testing period for our method is only 1 min, and the testing
230
frequency can be increased according to requirements, thus being a very rapid and
231
robust method. This method can be used into some site-specific works, such as water
232
quality online monitoring station.
233 234
Theoretical concentration of microcystins was calculated using following equation: CT =
235
C⋅V V + v⋅t
Eqn. 2
236
Where CT is the theoretical microcystins concentration value (μg/L); C is the
237
initial concentration in spiked water sample (μg/L); V is the initial volume of spiked
238
water sample (L); t is the time of water addition (min); v is the rate of water adding
239
(L/min).
240
The characterization of microcystins recovery was implemented to assess the
241
property of the detection method by comparing measured value with the theoretical
242
values (3 min, 6 min, 9 min, 12 min, 15 min, 18 min, 21 min, 24 min, 27 min). 11
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
243
Page 12 of 26
The recovery of microcystins was calculated using the following equation: R=
244
CM % CT
Eqn. 3
245
Where R is the recovery of proposed method (%); CM is the measured microcystins
246
concentration value (μg/L) and CT is the theoretical microcystins concentration.
247
Table 2 reports the recovery rates for the analyzed microcystins, which were in
248
the range of 77-103.6%, thus showing suitable sensitivity, precision, and accuracy for
249
microcystins detection.
250
In conclusion, in this study we propose a rapid and real time microcystins
251
determination method based on the use of paper spray ionization coupled to a
252
time-of-flight mass spectrometer. Rapid detection was performed to record the
253
release process of microcystins, and the identification time was reduced from several
254
hours to a few minutes. This method exhibits the potential to perform rapid, real
255
time and high-throughput assays for the detection and quantitation of microcystins,
256
and it would be of significant interest for environmental and food-safety applications.
257
The ability to rapidly collect quantitative and qualitative information may bridge the
258
gap in current methodologies which are either slow and accurate or fast and
259
nonspecific.
260
■ACKNOWLEDGEMENTS
261
This study is financially supported by “National Instrumentation Program of
262
China” (No. 2011YQ17006703, 2011YQ17006704) and “Special Support Program for
263
High-level Personnel of Guangdong” (2014TQ01X190). The authors would like to
264
express their appreciation to colleagues in Jinan University, Guangdong Provincial 12
ACS Paragon Plus Environment
Page 13 of 26
Journal of Agricultural and Food Chemistry
265
Engineering Research Center for On-line Source Apportionment System of Air
266
Pollution and Hexin Analytical Instrument Company.
267
■REFERENCES
268
(1) Bortolia, S; Oliveira-Silva, D.; Krüger, T; Dörra, F. A.; Colepicolo, P.; Volmer, D. A.;
269
Pinto, E. Growth and microcystin production of a Brazilian Microcystis aeruginosa
270
strain (LTPNA 02) under different nutrient conditions. Rev. Bras. Farmacogn. 2014, 24,
271
389-398.
272
(2) Wu, X. Q.; Wang, C. B.; Tian, C. C.; Xiao, B. D.; Song, L. R. Evaluation of the
273
potential of anoxic biodegradation of intracellular and dissolved microcystins in lake
274
sediments. J. Hazard. Mater. 2015, 286, 395–401.
275
(3) Chorus, I.; Falconer, I. R.; Salas, H. J.; Bartram J. Health risks caused by freshwater
276
cyanobacteria in recreational waters. J. Toxicol. Environ. Health, Part B. 2000, 3,
277
323–347.
278
(4) Ballot, A.; Sandvik, M.; Rundberget, T.; Botha, C. J.; Miles, C. O. Diversity of
279
cyanobacteria and cyanotoxins in Hartbeespoort Dam, South Africa. Mar. Freshwater
280
Res. 2014, 65, 175–189.
281
(5) Rodrigues, M. A.; Reis, M. P.; Mateus, M. C. Liquid chromatography/negative
282
electrospray ionization iontrap MS2 mass spectrometry application for the
283
determination of microcystins occurrence in southern Portugal water reservoirs.
284
Toxicon. 2013, 74, 8–18.
285
(6) Li, Y. W.; Zhan, X. J.; Xiang, L.; Deng, Z. S.; Huang, B. H.; Wen, H. F.; Sun, T. F.; Cai, Q.
286
Y.; Li, H.; Mo, C. H. Analysis of trace microcystins in vegetables using solid-phase 13
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
287
extraction followed by high performance liquid chromatography triple-quadrupole
288
mass spectrometry. J. Agric. Food Chem. 2014, 62, 11831-11839.
289
(7) Funari, E.; Testai, E. Human health risk assessment related to cyanotoxins
290
exposure. Crit. Rev. Toxicol. 2008, 38, 97−125.
291
(8) Yan, H.; Pan, G.; Zhang, M.; Chen, H.; Zou, H. Study on the extraction and
292
purification of microcystins. Acta Sci. Circumstantiae. 2004, 24,355−359.
293
(9) Van, D. F. M. Marine algal toxins: origins, health effects, and their increased
294
occurrence. Environ. Health Perspect. 2000, 108, 133–141.
295
(10) Maitham, A. A.; Kyle, D. H.; Daniel, D. S.; David, C. Methods for simultaneous
296
detection of the cyanotoxins BMAA, DABA, and anatoxin-a in environmental samples.
297
Toxicon. 2013, 76, 316–325.
298
(11) Preece, E. P.; Moore, B. C.; Swanson, M. E.; Hardy, F. J. Identifying best methods
299
for routine ELISA detection of microcystin in seafood. Environ. Monit. Assess. 2015,
300
187, 12-15.
301
(12) Wang, C.; Tian, C.; Tian, Y.; Feng, B.; We, S.; Li, Y.; Wu, X.; Xiao, B. A sensitive
302
method for the determination of total microcystins in water and sediment samples
303
by liquid chromatography with uorescence detection. Anal. Methods. 2015, 7,
304
759–765.
305
(13) Rivetti, C.; Gomez-Canela, C.; Lacorte, S.; Barata, C.; Liquid chromatography
306
coupled with tandem mass spectrometry to characterise trace levels of
307
cyanobacteria and dinoflagellate toxins in suspended solids and sediments. Anal.
308
Bioanal. Chem. 2015, 407, 1451–1462. 14
ACS Paragon Plus Environment
Page 14 of 26
Page 15 of 26
Journal of Agricultural and Food Chemistry
309
(14) Poon, K. F.; Lam, M. H. W.; Lam, P. K. S.; Wong, B. S. F. Determination of
310
microcystins
311
microextraction-high-performance liquid chromatography. Environ. Toxicol. Chem.
312
2001, 20, 1648-1655.
313
(15) Guo, X. C.; Xie, P.; Chen, J.; Tuo, X.; Deng, X. W.; Li, S. C.; Yu, D. Z.; Zeng, C.
314
Simultaneous quantitative determination of microcystin-LR and its glutathione
315
metabolites in rat liver by liquid chromatography–tandem mass spectrometry. J.
316
Chromatogr., B 2014, 963, 54–61.
317
(16) Mol, H. G. J.; van Dam, R. C. J.; Steijger, O. M. Determination of polar
318
organophosphorus pesticides in vegetables and fruits using liquid chromatography
319
with tandem mass spectrometry: selection of extraction solvent. J. Chromatogr., A
320
2003, 1015, 119−127.
321
(17) Singh, S.; Asthana, R. K. Assessment of microcystin concentration in carp and
322
catfish:a case study from Lakshmikund Pond, Varanasi, India. Bull. Environ. Contam.
323
Toxicol. 2014, 92, 687–692.
324
(18) Li, W.; Xie, P.; Chen, J.; He, J.; Guo, X. C.; Yu, D. Z.; Chen, L. Quantitative liquid
325
chromatography–tandem mass spectrometry method for determination of
326
microcystin-RR and its glutathione and cysteine conjugates in fish plasma and bile. J.
327
Chromatogr., B 2014, 963, 113–118.
328
(19) Espy, R. D.; Teunissen, S. F.; Manicke, N. E.; Ren, Y.; Ouyang, Z.; Asten A. V.; Cooks,
329
R. G. Paper spray and extraction spray mass spectrometry for the direct and
330
simultaneous quantification of eight drugs of abuse in whole blood. Anal. Chem.
in
cyanobacterial
blooms
15
ACS Paragon Plus Environment
by
solid-phase
Journal of Agricultural and Food Chemistry
331
2014, 86, 7712−7718.
332
(20) Chen, H. W.; Zheng, J.; Zhang, X.; Luo, M. B.; Wang, Z. C.; Qiao, X. L., Surface
333
desorption atmospheric pressure chemical ionization mass spectrometry for direct
334
ambient sample analysis without toxic chemical contamination. J. Mass Spectrom.
335
2007, 42, 1045-1056.
336
(21) Harris, G. A.; Fernandez, F. M. Simulations and experimental investigation of
337
atmospheric transport in an ambient metastable-induced chemical ionization source.
338
Anal. Chem. 2009, 81, 322-329.
339
(22) Wang, H.; Liu, J. J.; Cooks, R. G.; Ouyang, Z. Paper spray for direct analysis of
340
complex mixtures using mass spectrometry. Angew. Chem. Int. Ed. 2010, 49, 877
341
–880.
342
(23) Espy, R.D.; Muliadi, A.R.; Ouyang, Z.; Cooks, R.G. Spray mechanism in paper spray
343
ionization. Int. J. Mass Spectrom. 2012, 325, 167–171.
344
(24) Hamid, A. M.; Wei, P.; Jarmusch, A. K.; Pirro, V.; Cooks, R. G. Discrimination of
345
Candida species by paper spray mass spectrometry. Int. J. Mass Spectrom. 2015, 378,
346
288–293.
347
(25) Hamid, A. M.; Jarmusch, A. K.; Pirro, V.; Pincus, D. H.; Clay, B. G.; Gervasi, G.;
348
Cooks, R. G. Rapid discrimination of bacteria by paper spray mass spectrometry. Anal.
349
Chem. 2014, 86, 7500−7507.
350
(26) Pavagadhi, S.; Natera, S.; Roessner, U.; Balasubramanian, R. Insights into
351
lipidomic perturbations in zebra fish tissues upon exposure to Microcystin-LR and
352
Microcystin-RR. Environ. Sci. Technol. 2013, 47, 14376−14384. 16
ACS Paragon Plus Environment
Page 16 of 26
Page 17 of 26
Journal of Agricultural and Food Chemistry
353
(27) Yuan, M.; Namikoshi, M.; Otsuki, A.; Rinehart, K. L.; Sivonen, K.; Watanabe, M. F.
354
Low-energy collisionally activated decomposition and structural characterization of
355
cyclic heptapeptide microcystins by electrospray ionization mass spectrometry. J.
356
Mass Spectrom. 1999, 34, 33−43.
357
(28) Kaloudis, T.; Zervou, S. K.; Tsimeli, K.; Triantis, T. M.; Fotiou, T.; Hiskia, A.
358
Determination of microcystins and nodularin (cyanobacterial toxins) in water by
359
LC–MS/MS. Monitoring of Lake Marathonas, a water reservoir of Athens, Greece. J.
360
Hazard. Mater. 2013, 263, 105– 115.
361
(29) Faassen, E. J.; Lürling, M. Occurrence of the microcystins MC-LW and MC-LF in
362
dutch surface waters and their contribution to total microcystin toxicity. Mar. Drugs
363
2013, 11, 2643-2654.
364 365 366 367 368 369 370 371 372 373 374 17
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
375 376
FIGURE CAPTIONS:
377
Figure1 Structures of microcystins MC-RR, 1, and MC-LR, 2.
378
Figure2 Sampling sites at Helong reservoir and Tianlu Lake
379
Figure3 Standard curve of MC-LR (m/z 996.2) and MC-RR (m/z 520.1)
380
Figure4 Mass spectra of the taget microcystins (MC-LR and MC-RR)
381
Figure5 Mass spectra of environmental water samples. Sampling site 2, May 2016,
382
Sample underwent cell lysis procedure
383
Figure6 Measured concentration curves of MC-RR and MC-LR
384
18
ACS Paragon Plus Environment
Page 18 of 26
Page 19 of 26
Journal of Agricultural and Food Chemistry
Table 1 Concentrations of Total Microcystins RR and LR in Reservoirs Water Samples Total Microcystins Concentration (μg/L) Sampling site 1 2 3 4 5 a
May
June
RR
LR
Total
RR
LR
Total
6.8 8.1
3.1 5.8
9.9 13.9