A LC-MS-MS rapid and sensitive method for dissolved ...

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The aim of this work was to monitoring the presence of some comune microcystins like: MC-RR, MC-LR, MC-LA, MC-YR, MC-LW, MC-LF (figure 1), Nodularin ...
A LCLC-MS MS--MS rapid and sensitive method for dissolved and intracellular algal toxins’s analysis in Pertusillo lake Grazia Accoto, Eustachio Acito, Achille Palma ARPAB-Centro Ricerche di Metaponto, ss. Jonica 106 km 448.2, 75012 Metaponto (Matera) - Italy [email protected] INTRODUCTION The algal bloom due to cyanobacteria such as Microcystis, Anabaena, and Planktothrix, may cause serious environmental problems. Cyanobacteria, also called blue-green algae, can produce a large spectra of toxins that can have effects on human health and/or on aquatic living organisms. Blue-green algae generally grow in lakes, ponds, and slow-moving streams when the water is warm and enriched with nutrients like phosphorus or nitrogen. Many different species of blue-green algae occur in waters, but the most commonly detected include Anabaena sp., Aphanizomenon sp., Microcystis sp., and Planktothrix sp. It’s not always the same species that blooms in a given waterbody, and the dominant species present can change over the course of the season. Therefore, it’s necessary to have analytical techniques to detect compounds structurally very different from each other. The aim of this work was to monitoring the presence of some comune microcystins like: MC-RR, MC-LR, MC-LA, MC-YR, MC-LW, MC-LF (figure 1), Nodularin (figure 2) and Anatoxin A (figure 3) in Pertusillo lake. WHO has set a threshold value for human health for Microcystin extracellular at 1 μg/l as concentration limit for drinking waters. Toxins were detected with high performance liquid chromatography (HPLC) coupled with LC-MS-MS/ion trap. SPE-on line pre-concentrated method was used for improve analytical sensitive. The analytical method was validated with commercially available standards and robustness was verified using classical SPE off-line method. The LOQ of this method was 0.01 ug/l for each compound.

FIGURE 1. Structure of microcystins where blue and greeenen FIGURE 2. Structure of Nodularin structure indicate the position of variable aminoacids.

Compound Microcystin LR

cas

Microcystin RR

111755374

Microcystin YR

101064-48-6

Microcystin LW

157622-02-1

Microcystin LF

154037-70-4

Microcystin LA

96180-79-9

Nodularin

118399277

FIGURE 3. Structure of Anatoxin A.

SPE ON ON--LINE METHOD Sample preparation and on-line injection Cartridge STRATA-X 25um, on-line extraction Loading step: pass 70 ml of sample, pre-filtered on 0,2 um cellulose nitrate membrane filters, through the spe-on line cartridge (flow 5ml/min) (figure 4). Elution step: pass mobile phase through SPE cartrige and analitical coloumn (figure 4). Washing step: pass 70 ml of ultrapure water through the spe-on line cartridge (flow 5ml/min) (figure 4).

FIGURE 4. SPE-on line steps.

MRM transition 995,6>135 995,6>127 995,6>107 995,6>155 498>135 519,8>135 519,8>127 519,8>174 519,8>200 519,8>887 1045,5>135 1045,5>107 1045,5>127 1045>135 523>135 523>910 1025,5>135 1025,5>127 1025,5>213 1025,5>375 1025>1007 1025>873 986,5>135 986,5>213 986,5>852 986,5>478 910,5>776 910,5>402 910,5>374 910,5>759 910>402,5 910>776 825,5>135 825,5>227 825,5>163 825,5>107 166,1>131,1 166,1>149,1 512,5>135 512,5>127

MC-LR 1 MC-LR 2 MC-LR 3 MC-LR 4 MC-LR 5 MC-RR 1 MC-RR 2 MC-RR 3 MC-RR 4 MC-RR 5 MC-YR 1 MC-YR 2 MC-YR 3 MC-YR 4 MC-YR 5 MC-YR 6 MC-LW 1 MC-LW 2 MC-LW 3 MC-LW 4 MC-LW 5 MC-LW 6 MC-LF 1 MC-LF 2 MC-LF 3 MC-LF 4 MC-LA 1 MC-LA 2 MC-LA 3 MC-LA 4 MC-LA 5 MC-LA 6 Nodularin 1 Nodularin 2 Nodularin 3 Nodularin 4 Anatoxin a 1 Anatoxin a 2 MC_ADRR 1 MC_ADRR 2

MRM transition 995,6>135 995,6>127 995,6>107 995,6>155 498>135 519,8>135 519,8>127 519,8>174 519,8>200 519,8>887 1045,5>135 1045,5>107 1045,5>127 1045>135 523>135 523>910 1025,5>135 1025,5>127 1025,5>213 1025,5>375 1025>1007 1025>873 986,5>135 986,5>213 986,5>852 986,5>478 910,5>776 910,5>402 910,5>374 910,5>759 910>402,5 910>776 825,5>135 825,5>227 825,5>163 825,5>107 166,1>131,1 166,1>149,1 512,5>135 512,5>127

TABLE 1. MRM mode parameters for algal toxins

LC –MS/MS conditions and method validation

APPLICATIONS ON ENVIRONMENTAL WATER

LC: System : HP 1100 (Agilent)

In 2014 the water of the lake was monitored for quantifying algal toxin potentially content in the lake to verify the possible health risk.

Column : Zorbax C18 5μm 2.1x150mm column with a guard column Mobile phase : Water (A) containing 0.1 % of formic acid and acetonitrile (B) Gradient : 0min:95/0 A/B, 2min: 95/5, 12min:5/95 until 17min, 20min:95/5 Flow rate : 300 μl/min; Cycle time : 35 min

From the month of July 2014 until November 2014 it was applied this method for verifying the presence of algal toxins. Once collected, the water sample was frozen at -20 °C for overnight and then sonicated for 30 minutes.

The sample was first filtered through a 0.45 um membrane and then from 0.2 um to MS/MS System : 4000Qtrap (ABSciex) triple quadrupole coupled with release any intracellular toxins. Subsequently the sample was subjected to analysis online ion trap with electrospray interface in positive ionization. SPE-LC-MS-MS. Algal toxin’s concentration in Pertusillo lake was never above the LOQ MS/MS parameters : Ion Spray Voltage 5500V in positive ionization; source 500°C; concentration 0,01 ug/l. nebulizer gas 40 psi; desolvation gas 40 psi; with nitrogen as collision gas, cone and Other Scientific results illustrated that there was a negative and significant relationships collision voltage optimized for each compounds. between chlorophyll-a and logarithm chlorophyll-a with nitrate (Bbalali S. et al). Acquisition mode : The multiple reaction monitoring (MRM) mode was used with the In June of 2014 until August of 2014 there was a total absence of macronutrients and most abundant MS/MS transition for quantitation and other transitions for significantly increasing the concentration of chlorophyll-a (figure 5). confirmation of substance identity (table 1). Method reproducibility was estimated by making 0,5

16,00

Nitrates (NO3) mg/l

0,45

14,00

Chlorophyll a (ug/l)

0,4

12,00

Phosphates (P mg/l)

0,35 0,3

10,00 0,25

8,00 0,2

6,00

0,15 0,1

2,00

0,05

0,00

0

FIGURE 5. Nitrates, chlorophylla, phosphates distribution in Pertusillo lake.

VL3_luglio 2013 VL6_luglio 2013 VL2_luglio 2013 VL4_agosto 2013 VL1_settembre 2013 VL2_settembre 2013 VL3_settembre 2013 VL6_settembre 2013 VL1_ottobre 2013 VL2_ottobre 2013 VL6_ottobre 2013 VL2_dicembre 2013 VL4_dicembre 2013 VL6_dicembre 2013 VL2_gennaio 2014 VL4_gennaio 2014 VL1_febbraio 2014 VL2_febbraio 2014 VL5_febbraio 2014 VL1_marzo 2014 VL3_marzo 2014 VL5_marzo 2014 VL1_aprile 2014 VL3_aprile 2014 VL6_aprile 2014 VL2_maggio 2014 VL4_maggio 2014 VL6_maggio 2014 VL2_giugno 2014 VL4_giugno 2014 VL1_luglio 2014 VL2_luglio 2014 VL5_luglio 2014 VL1_agosto 2014 VL3_agosto 2014 VL5_agosto 2014

4,00

Phosphates (P mg/l)

Method robustness was verified using off-line SPE pre-concentration as described by Messineo et Al.

18,00 Nitrates (NO3 mg/l) e Chlorophyll (ug/l)

six consecutive 150ml injections water sample spiked with 0.01 μg/L of each analytical standard. Retention time and peak area reproducibilities show good precision.

REFERENCES • Guzzella L. et al. Dissolved and intracellular microcystins in lake water during Planktothriix rubescens algal bloom: HPLC quantification and crustacean acute toxicity test; IRSA-CNR poster ; on line: http://www.microbiotests.be/posters/posterSETAC%20microcistine%20finale.pdf • Messineo V. et al.; Cyanobacterial toxins in Italian freshwaters; Limnologia 39 (2009) 95-106. • Diane M. Orihel et al.; Internal nutrient loading may increase microcystin concentrations in freshwater lakes by promoting growth of Microcystis populations; Ann. Limnol. - Int. J. Lim. 49 (2013) 225–235. • Bbalali S. et al; Relationships between Nutrients and Chlorophyll a Concentration in the International Alma Gol Wetland, Iran; J Aquac Res Development 2013, 4:3.

Mediterranean Forum on Water Resources - Matera 18 18--22 Ottobre 2015

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