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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

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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


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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


Rapid

determination;

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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



67

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aquatic toxins occur per year, with a 1.5% mortality rate.9

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Microcystins have been continually studied mainly due to their toxicity to

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human and enhanced occurrence frequency. The monitoring of the quality of water

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bodies to assess the presence of microcystins is of the utmost importance and often

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difficult because cyanobacterial blooms may generate many different kinds of

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microcystins. 10 For successful treatment of microcystins and to minimize the risk to

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human health, sensitive and reliable methods able to detect the different types of

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microcystins are urgently required. Several methods for microcystins determination,

75

such as biological-based screening methods and analytical techniques, have been

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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.

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performance liquid chromatography (HPLC)12 and HPLC coupled to mass

85

spectrometry (HPLC-MS),13 are effective and powerful technologies to detect

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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


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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

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Ambient ionization methods, such as extractive electrospray ionization (EESI),

93

desorption electrospray ionization (DESI), and others, have been used for rapid

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analysis of complex analyte without sample preparation.19-21 The paper spray

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technique is an ambient ionization method in which sample storage, separation and

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ionization processes are completed on a piece of paper with a sharp point.22

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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

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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

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Instrumentation and Chemicals. All chemicals used in this study were analytical

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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

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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



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

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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

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the sample. Paper was cut into a equilateral triangle (height 11mm, base 10mm).

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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


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the range m/z 100-1500. The instrument was externally calibrated using PEG 1000>.

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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

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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



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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


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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



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


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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

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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



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The recovery of microcystins was calculated using the following equation: R=

244

CM % CT

Eqn. 3

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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.

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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.

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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


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Engineering Research Center for On-line Source Apportionment System of Air

266

Pollution and Hexin Analytical Instrument Company.

267

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(27) Yuan, M.; Namikoshi, M.; Otsuki, A.; Rinehart, K. L.; Sivonen, K.; Watanabe, M. F.

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Low-energy collisionally activated decomposition and structural characterization of

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cyclic heptapeptide microcystins by electrospray ionization mass spectrometry. J.

356

Mass Spectrom. 1999, 34, 33−43.

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(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

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2013, 11, 2643-2654.

364 365 366 367 368 369 370 371 372 373 374 17



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

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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

Rapid Microcystin Determination Using a Paper ... - ACS Publications

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