Rapid Microcystin Determination Using a Paper Spray Ionization

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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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Rapid Microcystins Determination Using a Paper Spray Ionization

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Method with Time-of-Flight Mass Spectrometry System

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†,‡ *, †, § †, § †, § †, § # 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])

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ABSTRACT: The eutrophication of surface water sources and climate changes have

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resulted in an annual explosion of cyanobacterial blooms in many irrigating and

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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.

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With this approach, direct analysis of microcystins mixtures without sample

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preparation has been achieved. Rapid detection was performed simulating the

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release process of microcystins in reservoirs water samples, and the routine

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detection frequency was every 3 minutes. The identification time of microcystins was

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reduced from several hours to few minutes. The limit of detection is 1 μg/L and the

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limit of quantitation is 3 μg/L. This method displays the ability for carrying out rapid,

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direct and high-throughput experiments for determination of microcystins, and it

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would be of significant interest for environmental and food safety applications.

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Microcystins;

38

KEYWORDS:

39

Simultaneous analysis;

Paper

spray

ionization;

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Rapid

determination;

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■INTRODUCTION For the past few years, with the industrial and agricultural development, water 1,2

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eutrophication is becoming more and more serious in China.

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eutrophication of surface water sources and climate changes have result in

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cyanobacterial blooms in many drinking and irrigating water resources.

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The aggravated

Microcystins are cyanotoxins with cyclic heptapeptide structures. The common

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structure

is

cyclo(D-Ala-X-D-MeAsp-Z-Adda-D-Glu-Mdha)

in

which

Adda

is

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(2S,3S,8S,9S)-3-amino-9-methoxy-2,6,8-trimethyl-10-phenyl-deca-4, 6-dienoic acid,

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Mdha is N-methyldehydroalanine, and D-MeAsp is D-erythromethylaspartic acid.

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Studies on the structure and identification of microcystins have been evolving from

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the early 1960s, identifying more than 90 variants of microcystins. Microcystin-LR

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(MC-LR) and microcystin-RR (MC-RR) are among the most common and harmful

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forms of cyanobacterial blooms in aquatic environments (Figure 1).6 Cropland can be

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polluted by irrigating with microcystin-contaminated water. These toxins can be

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accumulated in different organisms and crops and can easily reach human consumers.

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Previous studies have shown that the consumption of aquatic products, drinking

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water and crops are important exposure routes of microcystins to humans.6

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Microcystins have the function of liver toxicity and tumor promotion. They not only have a

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toxic effect on aquatic organisms, but also harmful to human health through drinking water

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and food chain. Microcystins are particularly prevalent in eutrophic waters following

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bloom lysis, with a wide range of concentrations (19−141 μg/L) in various water

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bodies throughout the world.

3-5

7

7,8

More than 60,000 human intoxications due to 3

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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,

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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

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and efficient approaches.

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There are two main approaches to detect the presence of microcystins:

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bio-chemical assays and instrument analytical methods. Biochemical assays such as

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invertebrate

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measurements, can detect the total amount of microcystins in samples. However,

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they are incapable of distinguishing microcystin analogues and may be strongly

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affected by matrix effects.

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

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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

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(ESI) source combined to LC has provided an easy mass detection method to ionize

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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

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need complex sample pretreatment in order to remove the reagents used and

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derivatization for HPLC analysis. 17,18

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

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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

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of microcystins in water. The identification of microcystins has been developed using

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a time-of-flight mass spectrometer system with paper spray ionization methods, and

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the identification time was reduced to a few minutes compared with HPLC/MS-based

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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

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and high-throughput assays for selective screening of microcystins.

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■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

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(Taoyuan, China) and dissolved in methanol. Chromatography paper(Grade 1) was

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bought from Whatman(Guangzhou, China).

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All experiments were performed with a homemade time-of-flight mass

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spectrometer (resolution>10,000 FWHM,FWHM means full width at half maximum, 5

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mass accuracy of 2 ppm,with in-source collision-induced dissociation function).

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Spectra were collected setting the instrument in positive mode.

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Environmental water samples

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Environmental water samples were collected from Helong reservoir and Tianlu Lake

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in Guangzhou, China (Figure 2). Sampling time was around May and June, 2016, the

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main season for cyanobacterial blooms. The top 15 cm of the water column was

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sampled, and stored in non-transparent glass bottles.

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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.

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Step 2: 500 mL of samples were transferred to a 1000 mL round flask. Samples

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were frozen and thawed 3 times by holding the flask alternately in a dry ice-acetone

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bath followed by a warm water bath (30 °C).

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Paper spray ionization and mass spectrometry. Paper spray ionization was used as

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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

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was 5 mm. The high voltage was applied by a copper clip. The sample was loaded

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onto the paper by using a dropper. Loaded sample was then air-dried, methanol was

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added and a voltage of 3.5 kV (DC) was applied via the copper clip. Sample was thus

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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

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function by using the following parameters: focus voltage, 132V; start voltage, 100V;

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end voltage, 70V; out-plate voltage, 93V; skimmer voltage, 35V.

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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

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in laboratory following these three steps:

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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;

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Step 3: Samples (5 parallel samples for each) were obtained at regular intervals

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(3 min) from the beaker. These samples were analyzed for the presence of

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microcystins. Detection time for one sample is only 1 min, therefore the detection

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frequency can be adjusted as required.

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■RESULTS AND DISCUSSION

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Method Development. The primary challenge in method development was to find a

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suitable set of conditions that would allow all microcystins to be analyzed

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simultaneously from a single sample. The method was optimized in terms of spray

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voltage, paper size and apex angle.

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Spray voltage should be as low as possible, under the premise of producing

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stable spray and ion current. Lower voltages are also desirable for lowering the 7

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background produced by chemical noise. Paper spray became completely unstable

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below 2.5 kV, thus 3.5 kV was used.

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Based on previous studies, the paper size and apex angle were investigated.

25

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The paper size has little effect on signal strength and stability, but it changes the

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amount of solvent that can be absorbed. Compared with paper size, the apex angle is

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a more important parameter, affecting the spectrum quality in a large extent. The

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apex angle influences the emission of charged droplets and the strength of the

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electric field as well as the competitive process of gaseous discharge. A more acute

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angle resulted in greater currents suggesting that the electric field strength exceeded

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the threshold required for stable electrospray and resulted in a gaseous discharge.

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According to experience, this kind of discharge will increase the complexity and noise,

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which has a negative effect on the quality of the spectrum. Therefore, a less acute

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angle of 50° was the better choice under the conditions tested.

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Method validation. The matrix effect (ME) is affected by many factors, such as

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the type of matrix, the treatment method and the instrument.6 The ME was

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evaluated by contrasting the calibration curves of microcystin standards in different

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solvent. The signal affect for microcystins was calculated using following equation:

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 S −S  ME = 100 ×  m s %  SS 

Eqn. 1

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The Sm represents the slopes of the matrix-matched calibration curves. Ss is the

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slopes of the solvent-only situation. The matrix effect can be ignored when ranging

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from −10% to +10%, it is considered minor when ranging from −20% to −10% or from

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+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

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microcystins were −9% to +12%.

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MC-RR and MC-LR standard solutions were used to assess the linearity and

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range of measurement 25. Seven standard solution concentrations (10, 20, 30, 40, 60,

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80, 100 μg/L) were analyzed to estimate the linearity and each point was detected

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three times. The regression lines of peak areas on different concentrations were

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obtained by least squares method (Figure 3). Estimated limits of detection (LODs)

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ranged from 1 to 5 μg/L. The LODs of MC-RR and MC-LR were confirmed by detecting

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spiked samples at the minimum concentration of 1 μg/L, with an S/N ratio >3. In the

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same way, the limits of quantitation (LOQs) are defined as 3 μg/L (S/N >10).

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Characterization of MC-LR and MC-RR. The molecular ion at m/z 996.2 [M+H]+ was

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observed in the mass spectrum of MC-LR (Figure 4), for the existing of arginine

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residue, in which the guanidine group decides the ionization state of target

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analytes.26 The double-charged molecular ion at m/z 520.1 [M+2H]2+ was observed in

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the spectrum of MC-RR, which contains two basic arginine residues (Figure 4).27

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The fragmentation of precursor ions of MC-LR and MC-RR gave product ions at

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m/z 135.2 and 213.3, respectively. The fragment ion at m/z 135.2 was recognized as

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the [phenyl-CH2−CH(OCH3)]+, formed by the a-cleavage at the methoxy group of the

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Adda β-amino acid moiety.28 The product ion at m/z 213.3 was recognized as

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[Glu-Mdha+H]+ produced by by cleavage of the N-methyldehydroalanine and α-linked

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glutamic acid. These findings confirm previously reported data. 25, 29

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Detection of microcystins in environmental water samples. Sufficient microcystins 9

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recovery cannot be Achieved without a cell lysis procedure, for microcystins are

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commonly membrane-bound or intracellular toxins in algae cells. For example, in log

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phase populations more than 90% of microcystins are intracellular. Total sample

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microcystins concentrations is an important index to quality guidelines for drinking

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water. Microcystins can be released into water if algal cells are destroyed.

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Environmental water samples, subjected to a cell lysis procedure, unspiked, were

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determined by the paper spray ionization method. In the positive mode, molecular

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ion at m/z 996.2 [M+H]+ was typical for MC-LR, while the double charge at m/z 520.1

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[M+2H]2+ was typical of MC-RR, showing a splitting in the isotope pattern of 0.5 m/z

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(Figure 5). If microcystins are present in a complex matrix, their detection is more

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difficult. However, paper spray ionization coupled to mass spectrometry. Is a robust

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approach based on molecular and fragment ions typical of microcystins that can be

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quickly detected. These results are consistent with our previous experiments and

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with other studies.19,23 Paper spray ionization is a particularly simple ambient

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ionization technique which can be employed to measure trace constituents of

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complex mixtures. In the analysis performed on the five sampling sites, the

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predominant toxin detected was MC-RR, with a range of concentration of 0-9.1 μg/L.

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MC-LR was detected in ranges from 0-5.8 μg/L (Table 1).

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Rapid and Simultaneous detection of microcystins in a complex mixture. The

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monitoring of the quality of water bodies to assess the presence of cyanotoxins is

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often difficult considering that cyanobacterial blooms may contain complex mixtures

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of microcystins. 10

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The Microcystins release process was simulated in the laboratory by spiking

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standard MC-LR and MC-RR in water samples. Microcystins concentration was

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determined with our method based on paper spray ionization and mass

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spectrometry and calculated using standard curves for samples collected every 3

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minutes. Using five parallel samples, standard deviation and standard error were

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calculated. In Figure 6 are reported measured concentrations for MC-RR and MC-LR.

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Both of the microcystins concentrations show a declining trend, with MC-RR

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concentration double that of MC-LR. Although the routine detection frequency is

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every 3 min, one whole testing period for our method is only 1 min, and the testing

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frequency can be increased according to requirements, thus being a very rapid and

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robust method. This method can be used into some site-specific works, such as water

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quality online monitoring station.

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Theoretical concentration of microcystins was calculated using following equation: CT =

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C⋅V V + v⋅t

Eqn. 2

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Where CT is the theoretical microcystins concentration value (μg/L); C is the

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initial concentration in spiked water sample (μg/L); V is the initial volume of spiked

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water sample (L); t is the time of water addition (min); v is the rate of water adding

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(L/min).

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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

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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=

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CM % CT

Eqn. 3

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Where R is the recovery of proposed method (%); CM is the measured microcystins

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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

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the range of 77-103.6%, thus showing suitable sensitivity, precision, and accuracy for

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microcystins detection.

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In conclusion, in this study we propose a rapid and real time microcystins

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determination method based on the use of paper spray ionization coupled to a

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time-of-flight mass spectrometer. Rapid detection was performed to record the

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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,

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and it would be of significant interest for environmental and food-safety applications.

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The ability to rapidly collect quantitative and qualitative information may bridge the

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gap in current methodologies which are either slow and accurate or fast and

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nonspecific.

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■ACKNOWLEDGEMENTS

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This study is financially supported by “National Instrumentation Program of

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China” (No. 2011YQ17006703, 2011YQ17006704) and “Special Support Program for

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High-level Personnel of Guangdong” (2014TQ01X190). The authors would like to

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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

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Pollution and Hexin Analytical Instrument Company.

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FIGURE CAPTIONS:

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Figure1 Structures of microcystins MC-RR, 1, and MC-LR, 2.

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Figure2 Sampling sites at Helong reservoir and Tianlu Lake

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Figure3 Standard curve of MC-LR (m/z 996.2) and MC-RR (m/z 520.1)

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Figure4 Mass spectra of the taget microcystins (MC-LR and MC-RR)

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Figure5 Mass spectra of environmental water samples. Sampling site 2, May 2016,

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Sample underwent cell lysis procedure

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Figure6 Measured concentration curves of MC-RR and MC-LR

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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