permanganate oxidation of microcystin-la: kinetics...

Research ArticlePermanganate Oxidation of Microcystin-LA KineticsQuantification and Implications for Effective DrinkingWater Treatment

David C Szlag 1 Brian Spies1 Regina G Szlag2 and Judy A Westrick2

1Department of Chemistry Oakland University Rochester MI 48309 USA2Department of Chemistry Wayne State University Detroit MI 48202 USA

Correspondence should be addressed to David C Szlag szlagoaklandedu

Received 22 October 2018 Accepted 17 March 2019 Published 28 May 2019

Academic Editor Valerio Matozzo

Copyright copy 2019 David C Szlag et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Permanganate pretreatment of drinking water is effective in transforming dissolved noxious contaminants and in reducinghalogenated by-products Permanganate targets specific compounds such as taste and odor compounds disinfection precursorsmanganese andnatural organic contaminants that are not removed readily by conventional treatment aloneCyanobacterial blooms(cHABs) can increase disinfection by-product precursors as well as the cyanotoxin microcystin (MC) a potent liver toxin MCtoxicity is conferred by a unique conserved amino acid Adda that inhibits protein phosphatase 1 and 2A Although over 150MC congeners have been reported thousands of MCs are statistically possible Over the last ten years one congener MC-LA hasbeen reported with increasing frequency making it one of the most common cyanotoxins identified in North American freshwatersystems yet its oxidation has not been widely studied Frequently Adda specific enzyme-linked immunosorbent assay (ELISA)and protein phosphatase inhibition assay (PPIA) are used to quantitate total MCs to evaluate treatment efficiency and exposureAnecdotal reports suggest that MC degradation products can cause interference with the Adda-ELISA MC-LA was used as themodel MC compound in this study PPIA quantitation of MC-LA in water agreed with liquid chromatography high resolutionmass spectrometry (LCHRMS) whereas the ELISA quantitation did not agree with LCHRMS quantitation We determined thesecond order rate constant for MC-LA as 118 plusmn 9 Mminus1 sminus1 the activation energy to be 212 kJ molminus1 and the rate to be independentof pH between pH 6 and 9 Ten oxidation products (OPs) were observed by LCHRMS and three primary reaction pathways areproposed The reaction pathways were used to explain differences in the quantification by Adda-ELISA HRMS and PPIA Theoxohydroxylation of MC-LA produced two major OPs C

46H67N7O14[M+H]+ = 9424819 and C

46H69N7O15[M+H]+ =9604925

Major OPs may contain an unmodified Adda and are the likely cause of interference with the Adda-ELISA Several governmentalagencies recommend the use of the Adda-ELISA to determine theMC quantitation for treatment efficiency and customer exposureyet our results suggest that these or other OPs interfere with the Adda-ELISA causing artificially high values for total MCs

1 Introduction

The severity and frequency of cyanobacteria blooms areincreasing due to a combination of factors including in-creased nitrogen and phosphorous pollution climate changeand altered system ecology which in some cases may be theresult of invasive species [1 2] Cyanobacteria blooms impactdrinking water treatment efficiency by increasing chemicaldemand and particulate load resulting in increased risk ofpathogen and cyanotoxin breakthrough [3 4] Microcystins(MCs) are the most common class of cyanotoxins During

log-growth phase of the bloom approximately 90 of themicrocystin is intracellular and 10 is extracellular Mosttreatment processes have specific targeted efficiencies for par-ticulate andor contaminant removal to remove MCs bothtypes of processes must be optimized Quantitative tools usedto monitor MC drinking water treatment removal efficiencyand exposure are critical for consumer safety Ideally thesetools ie assays would determine the concentration of MCsas well as the toxicity of the water and would be simpleenough to be performed at the treatment plant Our goal wasto vet the use of two commercialMC assays to quantitateMCs

HindawiJournal of ToxicologyVolume 2019 Article ID 3231473 13 pageshttpsdoiorg10115520193231473

2 Journal of Toxicology

HNN

NH

NH HN

HN

HN

OCOOH

COOH OOO

OO

O

Exact Mass 9094848

AB

C1

2

34

5

6

78

X=LEU 2

Y=ALA 4

ALA 1

MeAsp 3

GLU 6

Adda 5

Mdha 7

Microcystin X-Pos Y-PosMC-LA Leu AlaMC-LR Leu ArgMC-RR Arg Arg

（3

MC-LA 46（67712

Figure 1 General structure of MC-LA showing variable amino acid positions and the primary (alkene) sites subject to permanganate oxida-tion

during permanganate oxidation and compare their results tothe quantitation of MCs by LCHRMS Our hypothesis wasthat the quantitation by Adda-ELISA PPIA and LCHRMSwould agree

MCs are cyclic heptapeptides of varying amino acidcomposition with respect to two positions (ldquoXrdquo and ldquoYrdquo)that contain variable L-amino acids as shown in Figure 1MCs are comprised of three D-amino acids alanine (Ala)methylaspartic acid (MeAsp) and glutamic acid (Glu)(Figure 1) The structure also includes two unique aminoacids N-methyldehydroalanine (Mdha) and 3-amino-9-methoxy-26 8-trimethyl-10-phenyldeca-4(E)6(E)-dienoicacid (Adda) (Figure 1) The amino acid Adda is foundin position 5 of the peptide ring in all MCs and it isthis moiety that is responsible for the toxicity The MCsinhibit irreversibly protein phosphatase 1 (PP1) and proteinphosphatase 2A (PP2A) and act as hepatotoxins because theyare preferentially transported into liver cells The toxicity ofthe various congeners varies by two orders of magnitude withMC-LR and MC-LA noted as the most toxic [5] MC-LAis prevalent in several regional water sources but has notbeen reported historically as widely as MC-LR [6] Not manycomprehensive oxidative treatment studies have investigatedthe oxidation ofMC-LA even though it has been determinedto have one of the slowest oxidation rates with the mostwidely used oxidant chlorine [7]

The primary mode of human MC exposure is throughdrinking water Historically conventional water treatmentfacilities have been able to meet the World Health Organiza-tion (WHO) guidance value of 10 120583gL MC-LR [8] The USEPA has expanded monitoring from a single congener MC-LR to encompass all MC congeners referred to as total MCsIn 2015 US EPA released a two-tier drinking water healthadvisory (HA) based on age preschool at 03 120583gL total MCsand above preschool age at 16 120583gL total MCs [9] Infant andtoddlers ingest 5 timesmore liquid to bodymass than school-age children and adults hence the decrease from 16 120583gLfor school-aged children and adults to 03 120583gL total MCsfor preschool-aged children More countries will likely postsimilar guidance Consequently the drinking water industryis tasked to determine the total concentration of a class of

compounds containing many congeners at the sub-part-per-billion concentration a challenging task Understanding thevariability in oxidation kinetics of MCs and the oxidationpathways is needed so that drinking water treatment canbe optimized for MC removal and to minimize toxic OPsInterferences must be identified and quantified to ensureaccurate process monitoring

Two classes of commercial biochemical assays ELISA [10]and PPIA [11] have become standard technologies to quanti-tate total MCs for drinking water utilities These kits may usedifferent antibodies or enzymes but generally respond to allMC congeners albeit variably Several governmental agenciesrecommend the use of the Adda-ELISA in the mistakennotion that Adda alone confers toxicity To date Adda-ELISAquantitation has not been directly correlated to MC toxicityThe PPIA method is based on the enzymes which the MCsinhibit and its quantitative response has been correlatedto toxicity Some studies have observed that the total MCconcentrations derived from ELISA and PPIA are oftenhigher than the total MC concentration derived from LCMS[12ndash14] Specifically after chemical oxidation treatment theAdda-ELISA total MC concentration has been reported tobe higher than that reported by LCMS [15 16] There is aneed to determine which OPs are causing a response with theAdda-ELISA or PPIA and what their relative concentrationsare under different reaction conditions

Often permanganate is added at the source water intakeAnoxic source water may contain metals in a reduced stateand the initial permanganate demand comes from theiroxidation Oxidative pretreatment of water using perman-ganate is employed commonly to oxidize nuisance naturalcontaminants such as taste and odor compounds Perman-ganate will selectively oxidize alkenes and arguably doesnot rupture cells and release MCs at normal treatmentdosages and contact times as determined by LCMS [17ndash19]In the early literature the basic permanganate mechanismwas believed to be the production of vicinal dihydroxyl orcarbonyl products [20] More recent work by Kim et al [21]describes the permanganate oxidation progressing throughvicinal hydroxyl-carbonyl pathways more like ozone oxida-tion [22 23] rather than vicinal dihydroxy OPs produced via

Journal of Toxicology 3

chlorine oxidation [24] Because of its selective oxidationpermanganate treatment may be useful early in the treatmentprocess for degradation of MCs as part of a multibarrieroxidation process to achieve the new low concentrationguidance for total MCs

To date three drinking water utilities in United Stateshave posted ldquodo not drinkrdquo advisories because of MCsdetected in their finished drinking water Carroll TownshipOH (2013) ToledoOH (2014) and SalemOR (2018) reportedMCs concentrations above 10 120583gL total MC Because theyserve 400000+ residents the Toledo OH advisory was themost publicized The finished drinking water was reportedto be above 10 ugL total MCs as measured by Adda-ELISAbut these results were not validated by LCMSMS Since ldquodonot drinkrdquo advisories result in financial loss for businessesand loss of consumer confidence in the local water authorityidentifying potential interferences in analytical methods thatresult in false positives is as critical as identifying potentiallytoxic OPs

Source water MC concentrations often exceed 1 120583gLand it is not unusual for concentrations to range from5 to 20 120583gL total MCs Our research focuses on howpermanganate can be used as an effective treatment barrierand on quantification of MCs after treatment In orderto determine permanganate efficiency we determine howcongener chemical variation impacts oxidation rates how thecyanobacteria cell is impacted by permanganate oxidationand how variations in source water quality (pH and naturalorganic compound) affect permanganate oxidation On-sitequantitation is essential formonitoring the treatment processWe evaluated the Adda-ELISA and PPIA on-site methodsby comparing the purported rate constants and by deter-mining the oxidation reaction mechanism and pathways byLCHRMS Furthermore determining the oxidation pathwayprovides insight regarding residual toxicity of OPs

2 Materials and Methods

21 Microcystins To compare our procedures to other re-searchers MC-LR -LA and -RR were purchased from EnzoLife Sciences Inc For the majority of the oxidation exper-iments however MC-LA was purified from a Microcystisbloomscum harvested from Upper Klamath Lake OR in2015 MCs were released from the cells by three freezethawcycles Lysate was centrifuged in a swinging bucket thenvacuum filtered through a 47 mm 04 120583m glass fibre filterA crude extract was made by passing the lysate through a60 cm3 C18 solid phase extraction column (Thermo FisherScientific USA) discarding the effluent and making thecrude extract by eluting the bound material with 100 HPLCgrademethanol MC-LAwas separated from the crudeextract by high performance liquid chromatography witha photodiode array detector using a C18 semipreparativecolumn (Atlantis dC18 OBD Prep Column 100A 5120583m 10mmx 100 mm column Waters Corp) MC-LA was separatedbased on retention time and by UV spectrum The MC-LAwas validated by high resolutionmass spectrometry (HRMS)Concentrated MC stock solutions were prepared at 05mgmlin MS grade methanol Beerrsquos Law with a molar extinction

coefficient of 39800 Lsdotmolminus1sdotcmminus1 was used to determine theactual concentration of the MC-LA stock solution prior todilution for the batch experiments [25]

A four-place mini jar test apparatus was used for alllaboratory experiments with the paddle mixer set at 50 rpmA 200 mL sample of DI buffered (ammonium carbonate) orlake water was dispensed into each reaction chamber of themini-mixer The jar test unit was placed in a temperature-controlled cabinet to obtain the desired temperature 30minutes prior to the start of batch experiments to reachthermal equilibrium An intermediate stock solution of MCwas prepared in DI water from the standardized stock TheMC stock solution was pipetted into the reaction chamber toobtain an initial concentration of 50120583gMCL for kinetic testsPrior to the start of the experiment 10uL of 005M sodiumthiosulfate was preloaded into 14mL conical microfuge tubesthat would receive samples to minimize delays in quenchingthe reaction Prior to the addition of permanganate a controlsample of 10 mL was taken from each chamber (t = 0seconds) Stock permanganate solution was added to thereaction chamber to bring the concentration to the desired20 mgL MnO

4

minus and 10 mL aliquots were collected atspecified time intervals into the tubes preloaded with sodiumthiosulfate Generally the kinetic studies of MC-LA wereperformed at six time points 0 2 4 6 20 and 40 minutesTemperatures for the Arrhenius plot were 4 10 18 2530 and 35∘C Two temperatures 25 and 35∘C were runfor the experiments in which all three methods Adda-ELISA PPIA and LCHRMS were used to quantitate MCsThese samples were immediately capped and vortexed for6-10 seconds Samples were held on ice for the durationof the batch experiment then stored at 4∘C and analyzedwithin 48 hours Prior to analysis by LCTOF PPIA orELISA samples were centrifuged in a microfuge to pelletMnO2

The sodium thiosulfate quenching solution was preparedby dissolving 301g of sodium thiosulfate in 25mL of 18 M-1015840Ωdeionized water This stock solution was stored in an airtightamber bottle away from light The stock solution was diluted110 as needed for experiments

The enzyme-linked immunosorbent assays (ELISA) werepurchased from Abraxis Inc Calibration curves for MCswere quantitated using the response curve of MC-LR usingstandards 0 015 05 10 25 and 50 120583gL All samples werediluted 20-fold to bring them into the range of the standardcurve All samples and standards were run in duplicate Plateswere quantified using a BioTek Synergy H1M microplatereader A four-parameter curve fit was performed usingBioTek Gen5 software

Protein Phosphatase Inhibition Assay (PPIA) was devel-oped by Zeulab SL Zaragoza Spain and distributed in theUSA by Abraxis Inc Calibration curves for MCs range from025 ndash 25 120583gL All samples were diluted 20-fold to bringthemwithin the range of the standard curveThis test is basedon inhibition of protein phosphatase 2A All samples andstandards were run in duplicate Calibration curves for MCswere quantitated using the response curve ofMC-LR A four-parameter curve fit was performed using the BioTek Gen5software


Table 1 Summary of second order rate constants for MCs

MC-LA MC-LR MC-RR Temp pH Referencek (Mminus1 sminus1) k (Mminus1 sminus1) k (Mminus1 sminus1) ∘C118 plusmn 9 320 plusmn 20 467 (N=1) 18 plusmn 1 68 plusmn 02 This study160 plusmn 13 386 plusmn 20 520 plusmn 9 21 plusmn 1 72 Kim et al [21]170 408 23 plusmn 1 76 Ding et al [26]

290 23 plusmn 1 7 Jeong et al [20]357 plusmn 18 418 20 72 Rodrıguez et al [27]

469 plusmn 37 25 67 Chen et al [28]

LCMS analysis was performed by HPLC (Agilent 1200)with a ZORBAX Eclipse Plus C18 (id 21 times 50 mm18 mm) using gradient (10-100) conditions with 01formic acid and acetonitrile (flow rate 04 mL minminus1) AnAgilent 6520 quadrupole-time-of-flight (Q-TOF) was usedfor identification and quantitation MS conditions were asfollows ionization ESI nebulizer 50 psi(N

2) drying gas 10

L minminus1 350∘C (N2) Vcap 5000 V fragmentor 100 V scan

mz 100ndash1500 10000 transientsscan 10000 references mz1210509 and 9220098 and collision energy 15ndash30 eV

22 Pseudo-First-Order Kinetic Analysis The oxidation reac-tion rate of MC-LA by MnO

4

minus can be expressed as

r = k [MC-LA]x [MnO4

minus]y (1)

where r is the reaction rate k is the rate constant [MC-LA]and [MnO

4

minus] are concentrations and x and y are the reactionorders with respect to MC-LA and MnO

4

minus respectively [28]When the concentration of MnO

4

minus is excessive (1) can besimplified to (2) and (3)

r = k1015840 [MC]x (2)

where the product of the 2nd order rate constant andpermanganate concentration is effectively constant and k1015840 isknown as a pseudo-first-order rate constant

k1015840 = k [MnO4

minus]y (3)

This allows the use of the first-order integrated rate law andthe determination of a pseudo-first-order rate constant k1015840

ln( 119872119862119872119862119900) = minus1198961015840119905 (4)

The natural log of the ratio MC concentration divided bythe initial MC concentration was plotted versus time Theslope of this line is equal to the pseudo-first-order rateconstant The concentration of permanganate is present in20-fold excess relative to the MC-LA and its concentrationis effectively unchanged over the 40-minute experiment Thesecond order rate constant is calculated by dividing k1015840 by themolar concentration of permanganate

3 Results and Discussion

31 Oxidation Kinetics ofMC-LAwith Permanganate Chem-ical oxidation is considered an excellent drinking water

treatment barrier for MC such that America Water WorkAssociation has a web-based modeling program to assistdrinking water management to estimate the efficiencies ofoxidation removal by chlorine permanganate and ozone[29] This program uses published kinetics pH dependentkinetics and activation energies (temperature dependentkinetics) in their predictive empirical model The authorsof this tool have identified four reoccurring problems (1)only a limited number of MCs have been investigated (2)often temperature and pH dependent experiments are notperformed (3) the impact of NOM and cyanobacteria cellson oxidation kinetics is highly variable and (4) kineticsdetermined by LCMS are different from kinetics determinedbyAdda-ELISA Our investigation provided temperature andpH dependence data on a less examined MC (MC-LA) Byusing Adda-ELISA PPIA and LCHRMS to measure MCconcentrations we are able to show that OPs were likelybinding to the Adda-ELISA antibody Finally elucidation ofthe reaction mechanism by LCHRMS gives insight as towhich potential OP products interfere with Adda-ELISA

We observed second order rate constants 120 plusmn 9 Mminus1sdotsminus1 and 114 plusmn 8 Mminus1sdot sminus1 for our purified and commerciallyavailable MC-LA respectively with 20 ppm MnO

4

minus at pH68 and 18 plusmn 1∘C Combining experiments (N=9) yielded anaverage second order rate constant at these conditions of 118plusmn9 Mminus1sdot sminus1 This is approximately 25 lower than previouslyreported values by Kim et al [21] and Ding et al [26] Wealso conducted a limited number of experiments with MC-RR and MC-LR Our rate constant values for MC-LR andMC-RR are consistent and in the range observed by otherresearchers and follow the trendMC-RRgtMC-LR gtMC-LA(Table 1) Correcting for the temperature difference betweenour conditions and those reported by Kim et al [21] or Dinget al [26] would increase our values by eight percent Sampledata are shown in Figure 2

The activation energy (Ea) was determined by taking thenatural log of the Arrhenius equation and plotting ln k versus1T (Figure 3) The coefficient of determination (R2) for thevalues in this study (N=24) over the temperature range 4∘Cto 35∘Cwas 085The Ea was 212 plusmn 19 kJ molminus1 pH was 67 plusmn02 and the preexponential factor was 577962 Mminus1 sminus1 ThisEa value for MC-LA is comparable to the value 215 plusmn 06 kJmolminus1 reported by Kim et al [21]

We investigated the effect of pH on the rate constant overthe range from pH 6 to pH 95 (Figure 4) and found thatpH has a negligible effect on k between pH 6 and 85 but


y = -00016x -00524

minus25

minus2

minus15

minus1

minus05

00 200 400 600 800 1000 1200 1400

ln (C

Co)

Time (s)

２2= 09963

Figure 2 Example MC-LA pseudo-first-order oxidation with 20 ppm permanganate

y = -25458x + 13267２2= 08488

000325 00033 000335 00034 000345 00035 000355 00036 000365000321T (K)

0

1

2

3

4

5

6

ln(k

)

Figure 3 Arrhenius plot for MC-LA over the temperature range 4∘C to 35∘C

appears to drop off at pH 95 Often pH of the water during abloom is gt 85 and the reduction of oxidation rate will reducetreatment efficiency

Many groups have studied the effects of NOM and thetype of NOM that reacts with permanganate Jeong et al [21]show that fulvic and humic acids react with permanganatewhile polysaccharides and proteins are much less reactiveThe effect of Lake Erie water (Table 2) was evaluated on thereaction of permanganate and MC-LA Within experimentalerror there was no effect on the rate constant at 22∘C and35∘CWedid not completely characterize theNOMin the lakewater but the Lake Erie water is low in humic and fulvic acidsand higher in proteinaceous NOM [30] Permanganate reactsmore slowly with proteins or amino acids relative to alkenes

We further support this observation with experimentscontaining Microcystis cells (M viridis) as shown in Table 3

Table 2 Effect of natural lake water on the MC-LA second orderrate constant

Source 22∘C 35∘CFiltered Lake Water 118 plusmn 15 Mminus1secminus1 1445 Mminus1secminus1

DI 1135 plusmn 65 Mminus1secminus1 1385 plusmn 125 Mminus1secminus1

Table 3 The variation of the MC-LA second order rate constant inthe presence ofM viridis cells

0 cellsmL 31000 cellsmL 160000 cellsmL 160000 lysed1145 Mminus1secminus1 122 Mminus1secminus1 1143 Mminus1secminus1 128 Mminus1secminus1

There is no statistical change in the k value as the amountof cyanobacteria cells is increased Furthermore there is no


0

20

40

60

80

100

120

140

160

k (M

-1s-1

5 6 7 8 9 104pH

)

Figure 4 Variation of the MC-LA second order rate constant from pH 6 to 95

Table 4 Apparent rate constants determined by three methods at 25∘C and 35∘C

Initial ConcentrationCo (120583gL)

PermanganateConcentration

(ppm)

Temperature∘C

kPPIAMminus1secminus1

kMSMminus1secminus1

kELISAMminus1secminus1

50 MC-LA 20 25 plusmn 1 116 plusmn 4 100 plusmn 14 615 plusmn 550 MC-LA 20 35 plusmn 1 145 plusmn 14 1345 plusmn 10 82 plusmn 25

reduction in k when a cell lysate was added More studiesof this phenomenon with wild Microcystis strains should beperformed before general conclusions are made howeverThe M viridis cells used in this experiment have beengrown in culture for many years and do not exhibit colonialmorphology they primarily grow as single or double cellswithout a mucilaginous layer that is often observed in wildstrains

In general our observations are consistent with previousworks that indicates that permanganate has a low reactivitywith proteinaceous material and that at moderate dosagespermanganate is unlikely to lyse cyanobacteria cells

32 Comparison between the Quantitation and Kinetics byAdda-ELISA PPIA and LCHRMS Both the Adda-ELISAand PPIA can use a microplate reader format providingdrinking water utilities a readily available on-site platformversus the LCHRMS which is a costly instrument that needsa highly trained technician Several natural water studies havecompared the results from these three technologies [12ndash14]showing general agreement but noting that the Adda-ELISAoften reports higher concentrations than LCMS whereasthe PPIA reports lower concentrations than the LCMSLimited studies have investigated Adda-ELISA and PPIAMC quantitation of waters that have undergone chemicaloxidation with chlorine ozone and chloramines [15 16]These results suggest that OPs from chlorination ozone andchloramines react with the Adda-ELISA antibody and thePPIA

This work determined the apparent second order kineticsof the permanganate oxidation of MC-LA by Adda-ELISA

PPIA and LCHRMS (Table 4) at 25plusmn 1 C and 355plusmn 1 C Sincebuffered DI water was used for this study the permanganatedemand is based on MC-LA and only MC-LA OP productsare present The kPPIA and kMS at both temperatures arevery similar while the kELISA has decreased by about 40These data suggest that the MC-LA OPs from permanganateoxidation may be binding with the Adda-ELISA but not thePPIA

The permanganate OP pathways for MC-LR have beenelucidated by Kim [21] The primary paths were (1) oxi-dation at one of the three alkenes to make a hydroxylketone and (2) oxidative cleavage at the Adda diene orMdha OPs that may bind with Adda-ELISA include theoxidation products of the Mdha because the Adda remainsintact and the oxidative cleavage of the C4-C5 alkeneThis cleavage leaves the ring intact and produces (4S5SE)-5-methoxy-24-dimethyl-6-phenylhex-2-enal (MMPHA)andor the (4S5SE)-5-methoxy-24-dimethyl-6-phenylhex-2-enoic acid (MMPHH) MMPHH and MMPHA chemicalstructures are like Adda and may interfere with the Adda-ELISA

The Adda-ELISA and PPIA results can be normalizedby the corresponding LCHRMS concentration (Figure 5)Similar to the observations reported by Guo et al [15] theAdda-ELISA concentrations from time 0 to 480 seconds wereabout twice the concentration determined by the LCHRMSThis is becuaseMC-LA bindsmore efficiently to the antibodythan MC-LR for which the calibration curve is generatedThe time 0-minute sample is taken before the addition ofpermanganate for each experiment and diluted 20x (as areall samples) and serves as a control for each experimental


PPIAMSMSELISAMSMS

200 400 600 800 10000Reaction Time (seconds)

0

05

1

15

2

25

3

35

4

45

Ratio

to M

SMS

Figure 5 PPIA and Adda-ELISA normalized by LCHRMS show that the ratio of the response of the Adda-ELISA increases as the reactionprogresses

runThe starting concentration of each experiment is 50 120583gLMC-LA and is determined independently by measurementof the MC-LA stock solution using UV spectroscopy After6-10 minutes the MC-LA concentration determined by theAdda-ELISA is considerably larger than the LCHRMS Theratio between Adda-ELISA and LCHRMS increased from 2to 4 This change in ratio reflects the change in the secondorder rate kinetics from concentrations determined by Adda-ELISA at 61 Mminus1secminus1 versus the 100 Mminus1secminus1 determinedby LCHRMS These data suggest that after 480 seconds anOP(s) is at a high enough concentration to interfere with theAdda-ELISA It is reasonable to suspect that the OP(s) has anintact Adda MMPHA or MMPHH

The normalized PPIA results are close to 10 across theentire time interval Often researchers use the PPIA topredict MC toxicity The MC concentrations and kinetic ratedetermined by PPIA matched the concentrations and kineticrate determined by LCHRMS (Table 4) within experimentalerror The OPs formed do not appear to inhibit PP2Asuggesting the OPs are unlikely to be toxic Both the PP2Aand Adda-ELISA bind to the Adda group in the MC but theyappear to react differently to the MC-LAOPs suggesting thatthe OPs reacting with Adda-ELISA do not inhibit proteinphosphatase These results suggest that after permanganateoxidation OPs can result in an Adda-ELISA false positivethat may not be related to the toxicity of the water It shouldbe noted that the hepatotoxicity is also a function of thetransport of MCs by the organic anion transporter (celltransmembrane protein) into liver cells Consequently futurestudies of OPs should include the use of cell-based assays todetermine if OPs that do inhibit PP1 and PP2A are capable ofentering liver cells and exhibiting toxicity

33 Oxidation Products and Pathways Over the last couple ofdecades scientists have usedmass spectrometry to determinethe oxidation reaction pathways of MC-LR for a variety ofoxidants Understanding the pathway provides insight intothe detoxification of MCs Since the Adda group is the toxic

center of the MC the oxidation of Adda is critical in thedetoxificationinactivation ofMCs However biodegradationstudies have shown that ring opening is another mechanismof detoxification [31 32] Other studies have suggested thatthe kinetics determined by mass spectrometry (as shownabove) do not provide the same kinetics as the Adda-ELISA[15 16] yet no mechanism was provided to explain thedisagreement The most likely explanation is that the Addaremains intact after oxidation and binds to the antibody it isalso possible that the antibody is not that specific for Addaand a piece(s) of the MC OP cross-reacts with the Addaantibody Elucidation of the oxidation pathway(s) can provideinsight as to the source of the false positives and promotedesign of better monitoring strategies

Three previous studies proposed oxidation pathways forMC-LR with permanganate [20 21 26] No previous studyhas elucidated the pathway for MC-LA In the two earlierstudies dihydroxylation of the Adda and Mdha alkeneswas the proposed product pathway Dihydroxylation acrossthe alkene is the permanganate alkaline (base) reactionfrequently used by synthetic chemists Jeong et al [20] alsosuggested hydroxylation of the Adda benzene Kim et al[21] discounted these pathways as they did not detect thedihydroxy MC-LR intermediate products nor did they thinkit is likely that permanganate would form hydroxylated ben-zene OPs given extremely slow rates of reaction for benzyliccompounds and permanganate Their pathway consists of 17products formed via five reactions (1) alkene to hydroxyke-tone (2) alkene to aldehydes (3) aldehyde to carboxylic acid(4) amide to amine or (5) carboxylic acid They identifiedthree classes of primary products (1) hydroxyketone acrossone of the alkenes (2) oxidation cleavage to form aldehydesandor (3) ketones Secondary and tertiary oxidation pro-ceeds via transformation of aldehydes to carboxylic acids andhydrolysis of amide peptide bonds to an amine and carboxylicacid which opens the ring Furthermore their results impliedthat the 120572120573 unsaturated carbonyl was less reactive than thediene to permanganate oxidation


MCLAOP1OP9OP11OP13

10 20 30 40 50 600Reaction Time (minutes)

100

1000

10000

100000

1000000

log(

Peak

Are

a)

Figure 6 MC-LAmajor oxidation products formed via reaction with 20 ppm permanganate at 22∘C

In our pathway studies 1000 120583gL MC-LA rather than50 120583gL MC-LA was used with 20 mgL permanganate toassure generation of sufficient concentration of minor OPsthat could be detected by LCHRMS We also extended thereaction time from 40 to 60 minutes

We identified eight major and several minor OPs of MC-LA that are analogous to those identified in Kim et alrsquos[21] work on MC-LR using similar LCHRMS analysis Ourproposed oxidation pathways are analogous to the schemedeveloped by Kim et al [21] for MC-LR oxidation withpermanganate Our labelling scheme follows theirs albeitwith the analogous formulas and masses representing MC-LA OPs The OPrsquos chemical formula mass label retentiontime mass difference and proposed structure are reportedin Table 5 Three major OPs ie one site of oxidation wereformed within 2 minutes the oxidation of the Adda diene(Figure 1) to form hydroxy ketones and the oxidative cleavageof the C4-C5 alkene to form an aldehyde product Since thethree products appear at the first-time point (2 min) it is notknown if theseOPs are produced in parallel or in seriesTheseprimary oxidation products undergo further oxidation Theoxidation reactions at the Mdha 120572120573 unsaturated carbonylseem to occur after theAdda diene reacts because they appearonly at longer times

The relative abundance of primaryOPs fromour perman-ganate MC-LA oxidation experiments is shown in Table 6and Figure 6 The primary oxidation products we observedfor the reaction ofMC-LAwith permanganate were generallyanalogous to those observed by Kim et al [21] except for thealdehyde OP oxidized to the carboxylic acid We observed

MC-LA analogs to OP11 hydroxyketones forming at sites Aor B (Figure 1) and analogs for OP1 and OP9 forming byoxidative cleavage at sites A and C respectively ProductsOP1 and OP11 continued to increase in concentration overthe course of the one-hour reaction whereas OP9 peakedat 20 minutes and began to decline by 60 minutes We didnot observe dihydroxy MC-LA (C

46H69N7O14 [M+H]+ =

94449747) or the aldehyde formed by oxidative cleavage atsite B This study using MC-LA and all previous pathwaystudies withMC-LR agree that OP1 analogs form rapidly andare the major product Furthermore this aldehyde OP shouldbe nontoxic and is unlikely to react with the Adda-ELISA orthe PPIA given that the Adda is cleaved

Secondary OPs OP10 and OP13 appear at two minutesand OP13 is the major OP of MC-LA Both OPs are formedby hydrolysis of an amide bond If the HRMS response isrelatively constant across the products OP13 is the mostabundant OP produced over the course of our one-hourreaction time Kim et al [21] observed the same behavior Aninteresting deviation from Kim et alrsquos observations is that wedo not observe OP3 OP4 and OP7 within one hour [21]Thesecondary OPs react via amide hydrolysis and result in theformation of carboxylic acids and opening of the peptide ringIn the Kim et alrsquos study [21] OP7 (formed via oxidation of thealdehyde from precursor OP3 or OP6) was a major productalong with primary OP1 and OP11 The minor products inKim et alrsquos study were analogous to OP3 OP4 OP9 OP10and OP17 [21] Similarly we did not observe OP3 OP4 andOP17 while we did see OP5 and OP7 Our minor productswere OP2 OP8 OP10 OP12 and OP15 OP16 andOP8 can be


Table 5 Description of MC-LA permanganate OP

Description OP [M+H]+ OP Formula RT (ppm)Figure 1 MC-LA 9104921 C

46H67N7O12

104 -22

O

O

O

OO

OO

O

NH

NH

NHN

HNNH

NH

COOH

COOH

1 7103356 C31H47N7O12

895 05

O

OO

O

OO

OO

O

N

NH

NH

HN

HN NH

NH

COOH

COOH

2 7123154 C30H45N7O13

na na

O

O

O

OOO

O

N

OO

NH

NH

COOH

COOH

HN

HN

HO

NH

NH6 7423254 C

31H47N7O14

057 -18

O

OH

HO O

O

OOO

OO

O

NH

NH

COOH

COOH

NHN

HNNH

（2

8 7603360 C31H49N7O15

055 335

O

OO OOO

OO O

NH

NH

COOH

COOH

NHN

HN

NH

NH9 9124714 C

45H65N7O13

989 -217

O

OO

OO

OO O

NH

NH

COOH

COOH

NHN

HN

NH

NH

HO（3 11 9424819 C

46H67N7O14

961 173


Table 5 Continued

Description OP [M+H]+ OP Formula RT (ppm)O

OO

O

OO

OO

O

NH

NH

COOH

COOH

OH

N

HN

HN

NH

NH

（3 12 9444612 C45H66N7O15

911 321

O

O O

O

OO

OO

NH

NH

COOH

COOHOH

HONHN

HNNH

（3

（2

13 9604925 C46H69N7O15

893 -02

O

O

O

OOO

O

O

O

NH

NH

COOH

COOH

OH

OH

NHN

HNNH

（3

（2

14 9624718 C45H67N7O16

895 98

O

O OO

OO

OO O

NH

NH

N

COOH

COOH

HO

OH

HN

HNHN

NH

（3

15 9744718 C46H67N7O16

890 -037

O

OO

OO

O

O O

O

N

N

HNH

COOH

COOH

OH

HO

HOHN

HN

NH

（3

（2

16 9924823 C46H69N7O17

815 -153

referred to as a tertiary OPs because they have been oxidizedtwice and hydrolyzed once Oxidative cleavage to carbonylsand oxidation to hydroxyketone at the three alkenes are thefirst reactions to take place We can infer that hydrolysis ofthe amide peptide bond is fast only after the initial oxidationof the alkene

From these observations we can infer that the majorpathways for MC-LA transformation areMC-LA997888rarrOP11997888rarrOP13997888rarrP16 MC-LA997888rarrOP1=gtP6 and MC-LA997888rarrOP9=gtOP12 Many OPs have several possible structural isomersas well as stereoisomers OP9 and OP10 have intact Addaand are likely to be toxic or interfere with the Adda-ELISAkit They are not the major products however for MC-LAOP9 is produced in relatively modest amounts relative toOP1 OP11 and OP13 but is not the likely interferent withthe Adda-ELISA because it is converted to OP12 OP10 is

likely to interfere with Adda-ELISA but is produced in minoramounts Preliminary fragmentation studies confirm thatP10 has an intact Adda OP11 and OP13 have four possibleisomers OP11 and OP13 (Figure 7) with intact Adda arelikely to interact with the Adda-ELISA OP13 is likely toexhibit interaction with the Adda-ELISA but because of theopen ring will not inhibit PP1 or PP2a This is the mostlikely candidate for the interference with the Adda-ELISASubsequent work will attempt to isolate OP11 and P13 and testfor interference with the Adda-ELISA and toxicity

4 Conclusions

The results presented provide a better understanding of howpermanganate reduces MC toxicity and identifies severalpractical implications Chemical oxidation of MC with 20


Table6Prop

osed

MC-

LAOPanalogou

stothe17MC-

LROPidentifi

edby

Kim

etal[21]Only12

ofthe17prod

uctswe

reidentifi

edin

ourstudies

with

MC-

LA

MCL

AOP1

OP2

OP6

OP8

OP9

OP10

OP11

OP12

OP13

OP14

OP15

OP16

MZ

9104921

7103356

7123149

7423254

760336

9124714

9304819

9424819

9444612

9604925

9624718

9744718

9924823

Time(m)

Area

2189187

1504

00

01750

444

2746

02854

00

04

167826

4368

00

01834

100

7539

07296

00

06

151568

7547

00

03629

213

11012

012552

00

010

110928

9106

00

03723

206

12217

014983

00

020

40476

9296

00

04527

100

11981

01500

9314

00

608064

9951

581

2804

1309

1980

010257

891

14905

2028

1559

6939


（3

（3 （2

OO O

OO O

O

OO

O OO

O

O

OO

NH

NH

HN

HN

HN

HNHNH

HO

HOOH

COOH

COOH

COOHCOOH

NHN

HN

HN

N

N

Figure 7 Two OP isomers OP11 and OP13 with intact Adda that are likely interferents Note that OP13 is an open ring

mgL permanganate with sufficient contact times can be aneffective first barrier for the more hydrophilic MCs MC-RR and MC-LR that oxidize rapidly We determined thatMC-LA has much slower kinetics than previously reportedRegional difference in bloom ecology gives rise to differ-ent congener profiles Identification of blooms dominatedby MC-LA is essential to manage the treatment processWe tested permanganate oxidation with Lake Erie waterand different levels of cells The NOM and cells did notchange the rate constants and confirmed the selectivity ofthe permanganate Furthermore there was no evidence thatpermanganate lysed the cells at 20 mgL We also showedthat OP can interfere with the Adda-ELISA whereas thePPIA kit tracks the HRMS well Furthermore inhibitionof PP2A is significantly reduced implying toxicity is alsoreduced The most likely OP for interfering with the Adda-ELISA kit is OP13 an open ring OP with intact Adda orthe MMPHAMMPHHWe also show that MC-LR and MC-LA congeners give rise to different ratios of analogous OPsand that further research is necessary regarding the toxicityof individual OPs One significant area of concern is theeffect of higher pH Most cHAB events cause the pH of thesource water to rise and it is not uncommon to observesource water with pHs between 8 and 95 It is possible thatpHs at the higher range may slow the reaction and producea different set of OPs that could enhance interference andtoxicity

Data Availability

The data used to support the findings of this study areavailable from the corresponding author upon request

Conflicts of Interest

The authors declare that they have no conflicts of interest

Acknowledgments

Wewould like to thank the following organizations OaklandUniversity Summer Research in Chemistry Water ResearchFoundation 4647 and CURES (P30 ES020957) We wouldlike to acknowledgeDr JohnnaBirbeck Lumigen Instrument

Center Wayne State University for her mass spectrometryexpertise

References

[1] J M OrsquoNeil T W Davis M A Burford and C J Gobler ldquoTherise of harmful cyanobacteria blooms The potential roles ofeutrophication and climate changerdquoHarmful Algae vol 14 pp313ndash334 2012

[2] H W Paerl and V J Paul ldquoClimate change links to globalexpansion of harmful cyanobacteriardquo Water Research vol 46no 5 pp 1349ndash1363 2012

[3] J A Westrick and D Szlag ldquoA cyanotoxin primer for drinkingwater professionalsrdquo Journal - American Water Works Associa-tion vol 110 no 8 pp E1ndashE16 2018

[4] X He Y-L Liu A Conklin et al ldquoToxic cyanobacteria anddrinking water Impacts detection and treatmentrdquo HarmfulAlgae vol 54 pp 174ndash193 2016

[5] J R Schmidt S W Wilhelm and G L Boyer ldquoThe fate ofmicrocystins in the environment and challenges for monitor-ingrdquo Toxins vol 6 no 12 pp 3354ndash3387 2014

[6] K A Loftin J L Graham E D Hilborn et al ldquoCyanotoxinsin inland lakes of the United States Occurrence and potentialrecreational health risks in the EPA National Lakes Assessment2007rdquoHarmful Algae vol 56 pp 77ndash90 2016

[7] L Ho G Onstad U V Gunten S Rinck-Pfeiffer K Craig andG Newcombe ldquoDifferences in the chlorine reactivity of fourmicrocystin analoguesrdquoWater Research vol 40 no 6 pp 1200ndash1209 2006

[8] World Health Organization Cyanobacterial Toxins Microcys-tin-LR in Drinking Water Cyanobacterial Toxins Microcystin-LR in Drinking Water WHOSDEWSH030457 WorldHealth Organization Geneva Switzerland 2003

[9] US EPADrinking Water Health Advisory for the CyanobacterialMicrocystin Toxins EPA-820R15101 Office of Water 2015

[10] US EPA ldquoMethod 546 Determination to total microcystinsand nodularins in drinking water and ambient water by Adda-enzyme-linked immunosorbent assayrdquo httpswwwepagovsitesproductionfiles2016-09documentsmethod-546-deter-mination-total-microcystins-nodularins-drinking-water-am-bient-water-adda-enzyme-linked-immunosorbent-assaypdf2018

[11] T Triantis K Tsimeli T Kaloudis N Thanassoulias E LytrasandAHiskia ldquoDevelopment of an integrated laboratory system


for the monitoring of cyanotoxins in surface and drinkingwatersrdquo Toxicon vol 55 no 5 pp 979ndash989 2010

[12] T Kaloudis S-K Zervou K Tsimeli T M Triantis T Fotiouand A Hiskia ldquoDetermination of microcystins and nodularin(cyanobacterial toxins) in water by LC-MSMS Monitoring ofLake Marathonas a water reservoir of Athens Greecerdquo Journalof Hazardous Materials vol 263 pp 105ndash115 2013

[13] A J Foss and M T Aubel ldquoUsing the MMPB techniqueto confirm microcystin concentrations in water measured byELISA and HPLC (UV MS MSMS)rdquo Toxicon vol 104 pp 91ndash101 2015

[14] D O Mountfort P Holland and J Sprosen ldquoMethod fordetecting classes of microcystins by combination of proteinphosphatase inhibition assay and ELISA Comparison with LC-MSrdquo Toxicon vol 45 no 2 pp 199ndash206 2005

[15] Y C Guo A K Lee R S Yates S Liang and P A RochelleldquoAnalysis of microcystins in drinking water by ELISA andLCMSMSrdquo Journal - American Water Works Association vol109 no 3 pp 13ndash25 2017

[16] X He B D Stanford C Adams E J Rosenfeldt and E CWert ldquoVaried influence of microcystin structural difference onELISA cross-reactivity and chlorination efficiency of congenermixturesrdquoWater Research vol 126 pp 515ndash523 2017

[17] J Fan R Daly P Hobson L Ho and J Brookes ldquoImpact ofpotassium permanganate on cyanobacterial cell integrity andtoxin release and degradationrdquo Chemosphere vol 92 no 5 pp529ndash534 2013

[18] J Fan L Ho P Hobson and J Brookes ldquoEvaluating the effec-tiveness of copper sulphate chlorine potassium permanganatehydrogen peroxide and ozone on cyanobacterial cell integrityrdquoWater Research vol 47 no 14 pp 5153ndash5164 2013

[19] H Ou N Gao C Wei Y Deng and J Qiao ldquoImmediateand long-term impacts of potassium permanganate on pho-tosynthetic activity survival and microcystin-LR release riskof Microcystis aeruginosardquo Journal of Hazardous Materials vol219-220 pp 267ndash275 2012

[20] B Jeong M-S Oh H-M Park et al ldquoElimination ofmicrocystin-LR and residual Mn species using permanganateand powder activated carbon Oxidation products and path-waysrdquoWater Research vol 114 pp 189ndash199 2017

[21] M S Kim H-J Lee K-M Lee J Seo and C Lee ldquoOxida-tion of microcystins by permanganate PH and temperature-dependent kinetics effect of DOM characteristics and oxida-tion mechanism revisitedrdquo Environmental Science amp Technol-ogy vol 52 no 12 pp 7054ndash7063 2018

[22] J Chang Z-L Chen Z Wang et al ldquoOzonation degradation ofmicrocystin-LR in aqueous solution Intermediates byproductsand pathwaysrdquoWater Research vol 63 pp 52ndash61 2014

[23] H Miao F Qin G Tao et al ldquoDetoxification and S59 degra-dation of microcystin-LR and -RR by ozonationrdquoChemospherevol 79 no 4 pp 355ndash361 2010

[24] Y Zhang Y Shao N Gao W Chu and Z Sun ldquoRemovalof microcystin-LR by free chlorine Identify of transformationproducts and disinfection by-products formationrdquo ChemicalEngineering Journal vol 287 pp 189ndash195 2016

[25] ISO 2019 ldquoWater quality ndashDetermination of microcystins ndashMethod using solid phase extraction (SPE) and high perfor-mance liquid chromatography (HPLC) with ultraviolet (UV)detectionrdquo 2005

[26] J Ding H Shi T Timmons and C Adams ldquoRelease andremoval of microcystins from microcystis during oxidative-

physical- andUV-based disinfectionrdquo Journal of EnvironmentalEngineering vol 136 no 1 pp 2ndash11 2010

[27] E Rodrıguez M E Majado J Meriluoto and J L AceroldquoOxidation of microcystins by permanganate reaction kineticsand implications for water treatmentrdquo Water Research vol 41no 1 pp 102ndash110 2007

[28] X Chen B Xiao J Liu T Fang and X Xu ldquoKinetics of theoxidation of MCRR by potassium permanganaterdquo Toxicon vol45 no 7 pp 911ndash917 2005

[29] B D Stanford C Adams E J Rosenfeldt E Arevalo andA Reinert ldquoCyanoTOX Tools for managing cyanotoxins indrinking water treatment with chemical oxidantsrdquo JournalAmerican Water Works Association vol 108 no 12 pp 41ndash462016

[30] R M Cory T W Davis G J Dick et al ldquoSeasonal dynamicsin dissolved organic matter hydrogen peroxide and cyanobac-terial bloom in Lake Erierdquo Frontiers inMarine Science vol 3article no 54 pp 1ndash17 2016

[31] DG BourneG J Jones R L BlakeleyA JonesA PNegri andP Riddles ldquoEnzymatic pathway for the bacterial degradation ofthe cyanobacterial cyclic peptide toxinmicrocystin LRrdquoAppliedand Environmental Microbiology vol 62 no 11 pp 4086ndash40941996

[32] B M Gulledge J B Aggen and A R Chamberlin ldquoLinearizedand truncated microcystin analogues as inhibitors of proteinphosphatases 1 and 2Ardquo Bioorganic amp Medicinal ChemistryLetters vol 13 no 17 pp 2903ndash2906 2003

Medicinal ChemistryInternational Journal of

Hindawiwwwhindawicom Volume 2018

ToxicologyJournal of


PainResearch and TreatmentHindawiwwwhindawicom Volume 2018


Arthritis

Neurology Research International


StrokeResearch and TreatmentHindawiwwwhindawicom Volume 2018

Drug DeliveryJournal of



Advances in Pharmacological Sciences

Tropical MedicineJournal of


AddictionJournal of



BioMed Research International

Emergency Medicine InternationalHindawiwwwhindawicom Volume 2018


Anesthesiology Research and Practice

Journal of


Pharmaceutics

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Infectious Diseases and Medical Microbiology


Canadian Journal of


Autoimmune DiseasesScientica



MEDIATORSINFLAMMATION

of

Submit your manuscripts atwwwhindawicom


HNN

NH

NH HN

HN

HN

OCOOH

COOH OOO

OO

O

Exact Mass 9094848

AB

C1

2

34

5

6

78

X=LEU 2

Y=ALA 4

ALA 1

MeAsp 3

GLU 6

Adda 5

Mdha 7

Microcystin X-Pos Y-PosMC-LA Leu AlaMC-LR Leu ArgMC-RR Arg Arg

（3

MC-LA 46（67712

Figure 1 General structure of MC-LA showing variable amino acid positions and the primary (alkene) sites subject to permanganate oxida-tion

during permanganate oxidation and compare their results tothe quantitation of MCs by LCHRMS Our hypothesis wasthat the quantitation by Adda-ELISA PPIA and LCHRMSwould agree

MCs are cyclic heptapeptides of varying amino acidcomposition with respect to two positions (ldquoXrdquo and ldquoYrdquo)that contain variable L-amino acids as shown in Figure 1MCs are comprised of three D-amino acids alanine (Ala)methylaspartic acid (MeAsp) and glutamic acid (Glu)(Figure 1) The structure also includes two unique aminoacids N-methyldehydroalanine (Mdha) and 3-amino-9-methoxy-26 8-trimethyl-10-phenyldeca-4(E)6(E)-dienoicacid (Adda) (Figure 1) The amino acid Adda is foundin position 5 of the peptide ring in all MCs and it isthis moiety that is responsible for the toxicity The MCsinhibit irreversibly protein phosphatase 1 (PP1) and proteinphosphatase 2A (PP2A) and act as hepatotoxins because theyare preferentially transported into liver cells The toxicity ofthe various congeners varies by two orders of magnitude withMC-LR and MC-LA noted as the most toxic [5] MC-LAis prevalent in several regional water sources but has notbeen reported historically as widely as MC-LR [6] Not manycomprehensive oxidative treatment studies have investigatedthe oxidation ofMC-LA even though it has been determinedto have one of the slowest oxidation rates with the mostwidely used oxidant chlorine [7]

The primary mode of human MC exposure is throughdrinking water Historically conventional water treatmentfacilities have been able to meet the World Health Organiza-tion (WHO) guidance value of 10 120583gL MC-LR [8] The USEPA has expanded monitoring from a single congener MC-LR to encompass all MC congeners referred to as total MCsIn 2015 US EPA released a two-tier drinking water healthadvisory (HA) based on age preschool at 03 120583gL total MCsand above preschool age at 16 120583gL total MCs [9] Infant andtoddlers ingest 5 timesmore liquid to bodymass than school-age children and adults hence the decrease from 16 120583gLfor school-aged children and adults to 03 120583gL total MCsfor preschool-aged children More countries will likely postsimilar guidance Consequently the drinking water industryis tasked to determine the total concentration of a class of

compounds containing many congeners at the sub-part-per-billion concentration a challenging task Understanding thevariability in oxidation kinetics of MCs and the oxidationpathways is needed so that drinking water treatment canbe optimized for MC removal and to minimize toxic OPsInterferences must be identified and quantified to ensureaccurate process monitoring

Two classes of commercial biochemical assays ELISA [10]and PPIA [11] have become standard technologies to quanti-tate total MCs for drinking water utilities These kits may usedifferent antibodies or enzymes but generally respond to allMC congeners albeit variably Several governmental agenciesrecommend the use of the Adda-ELISA in the mistakennotion that Adda alone confers toxicity To date Adda-ELISAquantitation has not been directly correlated to MC toxicityThe PPIA method is based on the enzymes which the MCsinhibit and its quantitative response has been correlatedto toxicity Some studies have observed that the total MCconcentrations derived from ELISA and PPIA are oftenhigher than the total MC concentration derived from LCMS[12ndash14] Specifically after chemical oxidation treatment theAdda-ELISA total MC concentration has been reported tobe higher than that reported by LCMS [15 16] There is aneed to determine which OPs are causing a response with theAdda-ELISA or PPIA and what their relative concentrationsare under different reaction conditions

Often permanganate is added at the source water intakeAnoxic source water may contain metals in a reduced stateand the initial permanganate demand comes from theiroxidation Oxidative pretreatment of water using perman-ganate is employed commonly to oxidize nuisance naturalcontaminants such as taste and odor compounds Perman-ganate will selectively oxidize alkenes and arguably doesnot rupture cells and release MCs at normal treatmentdosages and contact times as determined by LCMS [17ndash19]In the early literature the basic permanganate mechanismwas believed to be the production of vicinal dihydroxyl orcarbonyl products [20] More recent work by Kim et al [21]describes the permanganate oxidation progressing throughvicinal hydroxyl-carbonyl pathways more like ozone oxida-tion [22 23] rather than vicinal dihydroxy OPs produced via









4











2) drying gas 10




4



minus]y (1)


4


4


4


r = k1015840 [MC]x (2)


k1015840 = k [MnO4

minus]y (3)


ln( 119872119862119872119862119900) = minus1198961015840119905 (4)






4





y = -00016x -00524

minus25

minus2

minus15

minus1

minus05

00 200 400 600 800 1000 1200 1400

ln (C

Co)

Time (s)

２2= 09963


y = -25458x + 13267２2= 08488

000325 00033 000335 00034 000345 00035 000355 00036 000365000321T (K)

0

1

2

3

4

5

6

ln(k

)












0

20

40

60

80

100

120

140

160

k (M

-1s-1

5 6 7 8 9 104pH

)





(ppm)

Temperature∘C


kMSMminus1secminus1











PPIAMSMSELISAMSMS


0

05

1

15

2

25

3

35

4

45

Ratio

to M

SMS








MCLAOP1OP9OP11OP13


100

1000

10000

100000

1000000

log(

Peak

Are

a)






46H69N7O14 [M+H]+ =






46H67N7O12

104 -22

O

O

O

OO

OO

O

NH

NH

NHN

HNNH

NH

COOH

COOH

1 7103356 C31H47N7O12

895 05

O

OO

O

OO

OO

O

N

NH

NH

HN

HN NH

NH

COOH

COOH

2 7123154 C30H45N7O13

na na

O

O

O

OOO

O

N

OO

NH

NH

COOH

COOH

HN

HN

HO

NH

NH6 7423254 C

31H47N7O14

057 -18

O

OH

HO O

O

OOO

OO

O

NH

NH

COOH

COOH

NHN

HNNH

（2

8 7603360 C31H49N7O15

055 335

O

OO OOO

OO O

NH

NH

COOH

COOH

NHN

HN

NH

NH9 9124714 C

45H65N7O13

989 -217

O

OO

OO

OO O

NH

NH

COOH

COOH

NHN

HN

NH

NH

HO（3 11 9424819 C

46H67N7O14

961 173


Table 5 Continued


OO

O

OO

OO

O

NH

NH

COOH

COOH

OH

N

HN

HN

NH

NH

（3 12 9444612 C45H66N7O15

911 321

O

O O

O

OO

OO

NH

NH

COOH

COOHOH

HONHN

HNNH

（3

（2

13 9604925 C46H69N7O15

893 -02

O

O

O

OOO

O

O

O

NH

NH

COOH

COOH

OH

OH

NHN

HNNH

（3

（2

14 9624718 C45H67N7O16

895 98

O

O OO

OO

OO O

NH

NH

N

COOH

COOH

HO

OH

HN

HNHN

NH

（3

15 9744718 C46H67N7O16

890 -037

O

OO

OO

O

O O

O

N

N

HNH

COOH

COOH

OH

HO

HOHN

HN

NH

（3

（2

16 9924823 C46H69N7O17

815 -153




4 Conclusions



Table6Prop

osed

MC-

LAOPanalogou

stothe17MC-

LROPidentifi

edby

Kim

etal[21]Only12

ofthe17prod

uctswe

reidentifi

edin

ourstudies

with

MC-

LA

MCL

AOP1

OP2

OP6

OP8

OP9

OP10

OP11

OP12

OP13

OP14

OP15

OP16

MZ

9104921

7103356

7123149

7423254

760336

9124714

9304819

9424819

9444612

9604925

9624718

9744718

9924823

Time(m)

Area

2189187

1504

00

01750

444

2746

02854

00

04

167826

4368

00

01834

100

7539

07296

00

06

151568

7547

00

03629

213

11012

012552

00

010

110928

9106

00

03723

206

12217

014983

00

020

40476

9296

00

04527

100

11981

01500

9314

00

608064

9951

581

2804

1309

1980

010257

891

14905

2028

1559

6939


（3

（3 （2

OO O

OO O

O

OO

O OO

O

O

OO

NH

NH

HN

HN

HN

HNHNH

HO

HOOH

COOH

COOH

COOHCOOH

NHN

HN

HN

N

N



Data Availability




Acknowledgments



References










































Arthritis










AddictionJournal of







Journal of


Pharmaceutics



Volume 2018



Canadian Journal of






of










4











2) drying gas 10




4



minus]y (1)


4


4


4


r = k1015840 [MC]x (2)


k1015840 = k [MnO4

minus]y (3)


ln( 119872119862119872119862119900) = minus1198961015840119905 (4)






4





y = -00016x -00524

minus25

minus2

minus15

minus1

minus05

00 200 400 600 800 1000 1200 1400

ln (C

Co)

Time (s)

２2= 09963


y = -25458x + 13267２2= 08488

000325 00033 000335 00034 000345 00035 000355 00036 000365000321T (K)

0

1

2

3

4

5

6

ln(k

)












0

20

40

60

80

100

120

140

160

k (M

-1s-1

5 6 7 8 9 104pH

)





(ppm)

Temperature∘C


kMSMminus1secminus1











PPIAMSMSELISAMSMS


0

05

1

15

2

25

3

35

4

45

Ratio

to M

SMS








MCLAOP1OP9OP11OP13


100

1000

10000

100000

1000000

log(

Peak

Are

a)






46H69N7O14 [M+H]+ =






46H67N7O12

104 -22

O

O

O

OO

OO

O

NH

NH

NHN

HNNH

NH

COOH

COOH

1 7103356 C31H47N7O12

895 05

O

OO

O

OO

OO

O

N

NH

NH

HN

HN NH

NH

COOH

COOH

2 7123154 C30H45N7O13

na na

O

O

O

OOO

O

N

OO

NH

NH

COOH

COOH

HN

HN

HO

NH

NH6 7423254 C

31H47N7O14

057 -18

O

OH

HO O

O

OOO

OO

O

NH

NH

COOH

COOH

NHN

HNNH

（2

8 7603360 C31H49N7O15

055 335

O

OO OOO

OO O

NH

NH

COOH

COOH

NHN

HN

NH

NH9 9124714 C

45H65N7O13

989 -217

O

OO

OO

OO O

NH

NH

COOH

COOH

NHN

HN

NH

NH

HO（3 11 9424819 C

46H67N7O14

961 173


Table 5 Continued


OO

O

OO

OO

O

NH

NH

COOH

COOH

OH

N

HN

HN

NH

NH

（3 12 9444612 C45H66N7O15

911 321

O

O O

O

OO

OO

NH

NH

COOH

COOHOH

HONHN

HNNH

（3

（2

13 9604925 C46H69N7O15

893 -02

O

O

O

OOO

O

O

O

NH

NH

COOH

COOH

OH

OH

NHN

HNNH

（3

（2

14 9624718 C45H67N7O16

895 98

O

O OO

OO

OO O

NH

NH

N

COOH

COOH

HO

OH

HN

HNHN

NH

（3

15 9744718 C46H67N7O16

890 -037

O

OO

OO

O

O O

O

N

N

HNH

COOH

COOH

OH

HO

HOHN

HN

NH

（3

（2

16 9924823 C46H69N7O17

815 -153




4 Conclusions



Table6Prop

osed

MC-

LAOPanalogou

stothe17MC-

LROPidentifi

edby

Kim

etal[21]Only12

ofthe17prod

uctswe

reidentifi

edin

ourstudies

with

MC-

LA

MCL

AOP1

OP2

OP6

OP8

OP9

OP10

OP11

OP12

OP13

OP14

OP15

OP16

MZ

9104921

7103356

7123149

7423254

760336

9124714

9304819

9424819

9444612

9604925

9624718

9744718

9924823

Time(m)

Area

2189187

1504

00

01750

444

2746

02854

00

04

167826

4368

00

01834

100

7539

07296

00

06

151568

7547

00

03629

213

11012

012552

00

010

110928

9106

00

03723

206

12217

014983

00

020

40476

9296

00

04527

100

11981

01500

9314

00

608064

9951

581

2804

1309

1980

010257

891

14905

2028

1559

6939


（3

（3 （2

OO O

OO O

O

OO

O OO

O

O

OO

NH

NH

HN

HN

HN

HNHNH

HO

HOOH

COOH

COOH

COOHCOOH

NHN

HN

HN

N

N



Data Availability




Acknowledgments



References










































Arthritis










AddictionJournal of







Journal of


Pharmaceutics



Volume 2018



Canadian Journal of






of








2) drying gas 10




4



minus]y (1)


4


4


4


r = k1015840 [MC]x (2)


k1015840 = k [MnO4

minus]y (3)


ln( 119872119862119872119862119900) = minus1198961015840119905 (4)






4





y = -00016x -00524

minus25

minus2

minus15

minus1

minus05

00 200 400 600 800 1000 1200 1400

ln (C

Co)

Time (s)

２2= 09963


y = -25458x + 13267２2= 08488

000325 00033 000335 00034 000345 00035 000355 00036 000365000321T (K)

0

1

2

3

4

5

6

ln(k

)












0

20

40

60

80

100

120

140

160

k (M

-1s-1

5 6 7 8 9 104pH

)





(ppm)

Temperature∘C


kMSMminus1secminus1











PPIAMSMSELISAMSMS


0

05

1

15

2

25

3

35

4

45

Ratio

to M

SMS








MCLAOP1OP9OP11OP13


100

1000

10000

100000

1000000

log(

Peak

Are

a)






46H69N7O14 [M+H]+ =






46H67N7O12

104 -22

O

O

O

OO

OO

O

NH

NH

NHN

HNNH

NH

COOH

COOH

1 7103356 C31H47N7O12

895 05

O

OO

O

OO

OO

O

N

NH

NH

HN

HN NH

NH

COOH

COOH

2 7123154 C30H45N7O13

na na

O

O

O

OOO

O

N

OO

NH

NH

COOH

COOH

HN

HN

HO

NH

NH6 7423254 C

31H47N7O14

057 -18

O

OH

HO O

O

OOO

OO

O

NH

NH

COOH

COOH

NHN

HNNH

（2

8 7603360 C31H49N7O15

055 335

O

OO OOO

OO O

NH

NH

COOH

COOH

NHN

HN

NH

NH9 9124714 C

45H65N7O13

989 -217

O

OO

OO

OO O

NH

NH

COOH

COOH

NHN

HN

NH

NH

HO（3 11 9424819 C

46H67N7O14

961 173


Table 5 Continued


OO

O

OO

OO

O

NH

NH

COOH

COOH

OH

N

HN

HN

NH

NH

（3 12 9444612 C45H66N7O15

911 321

O

O O

O

OO

OO

NH

NH

COOH

COOHOH

HONHN

HNNH

（3

（2

13 9604925 C46H69N7O15

893 -02

O

O

O

OOO

O

O

O

NH

NH

COOH

COOH

OH

OH

NHN

HNNH

（3

（2

14 9624718 C45H67N7O16

895 98

O

O OO

OO

OO O

NH

NH

N

COOH

COOH

HO

OH

HN

HNHN

NH

（3

15 9744718 C46H67N7O16

890 -037

O

OO

OO

O

O O

O

N

N

HNH

COOH

COOH

OH

HO

HOHN

HN

NH

（3

（2

16 9924823 C46H69N7O17

815 -153




4 Conclusions



Table6Prop

osed

MC-

LAOPanalogou

stothe17MC-

LROPidentifi

edby

Kim

etal[21]Only12

ofthe17prod

uctswe

reidentifi

edin

ourstudies

with

MC-

LA

MCL

AOP1

OP2

OP6

OP8

OP9

OP10

OP11

OP12

OP13

OP14

OP15

OP16

MZ

9104921

7103356

7123149

7423254

760336

9124714

9304819

9424819

9444612

9604925

9624718

9744718

9924823

Time(m)

Area

2189187

1504

00

01750

444

2746

02854

00

04

167826

4368

00

01834

100

7539

07296

00

06

151568

7547

00

03629

213

11012

012552

00

010

110928

9106

00

03723

206

12217

014983

00

020

40476

9296

00

04527

100

11981

01500

9314

00

608064

9951

581

2804

1309

1980

010257

891

14905

2028

1559

6939


（3

（3 （2

OO O

OO O

O

OO

O OO

O

O

OO

NH

NH

HN

HN

HN

HNHNH

HO

HOOH

COOH

COOH

COOHCOOH

NHN

HN

HN

N

N



Data Availability




Acknowledgments



References










































Arthritis










AddictionJournal of







Journal of


Pharmaceutics



Volume 2018



Canadian Journal of






of



y = -00016x -00524

minus25

minus2

minus15

minus1

minus05

00 200 400 600 800 1000 1200 1400

ln (C

Co)

Time (s)

２2= 09963


y = -25458x + 13267２2= 08488

000325 00033 000335 00034 000345 00035 000355 00036 000365000321T (K)

0

1

2

3

4

5

6

ln(k

)












0

20

40

60

80

100

120

140

160

k (M

-1s-1

5 6 7 8 9 104pH

)





(ppm)

Temperature∘C


kMSMminus1secminus1











PPIAMSMSELISAMSMS


0

05

1

15

2

25

3

35

4

45

Ratio

to M

SMS








MCLAOP1OP9OP11OP13


100

1000

10000

100000

1000000

log(

Peak

Are

a)






46H69N7O14 [M+H]+ =






46H67N7O12

104 -22

O

O

O

OO

OO

O

NH

NH

NHN

HNNH

NH

COOH

COOH

1 7103356 C31H47N7O12

895 05

O

OO

O

OO

OO

O

N

NH

NH

HN

HN NH

NH

COOH

COOH

2 7123154 C30H45N7O13

na na

O

O

O

OOO

O

N

OO

NH

NH

COOH

COOH

HN

HN

HO

NH

NH6 7423254 C

31H47N7O14

057 -18

O

OH

HO O

O

OOO

OO

O

NH

NH

COOH

COOH

NHN

HNNH

（2

8 7603360 C31H49N7O15

055 335

O

OO OOO

OO O

NH

NH

COOH

COOH

NHN

HN

NH

NH9 9124714 C

45H65N7O13

989 -217

O

OO

OO

OO O

NH

NH

COOH

COOH

NHN

HN

NH

NH

HO（3 11 9424819 C

46H67N7O14

961 173


Table 5 Continued


OO

O

OO

OO

O

NH

NH

COOH

COOH

OH

N

HN

HN

NH

NH

（3 12 9444612 C45H66N7O15

911 321

O

O O

O

OO

OO

NH

NH

COOH

COOHOH

HONHN

HNNH

（3

（2

13 9604925 C46H69N7O15

893 -02

O

O

O

OOO

O

O

O

NH

NH

COOH

COOH

OH

OH

NHN

HNNH

（3

（2

14 9624718 C45H67N7O16

895 98

O

O OO

OO

OO O

NH

NH

N

COOH

COOH

HO

OH

HN

HNHN

NH

（3

15 9744718 C46H67N7O16

890 -037

O

OO

OO

O

O O

O

N

N

HNH

COOH

COOH

OH

HO

HOHN

HN

NH

（3

（2

16 9924823 C46H69N7O17

815 -153




4 Conclusions



Table6Prop

osed

MC-

LAOPanalogou

stothe17MC-

LROPidentifi

edby

Kim

etal[21]Only12

ofthe17prod

uctswe

reidentifi

edin

ourstudies

with

MC-

LA

MCL

AOP1

OP2

OP6

OP8

OP9

OP10

OP11

OP12

OP13

OP14

OP15

OP16

MZ

9104921

7103356

7123149

7423254

760336

9124714

9304819

9424819

9444612

9604925

9624718

9744718

9924823

Time(m)

Area

2189187

1504

00

01750

444

2746

02854

00

04

167826

4368

00

01834

100

7539

07296

00

06

151568

7547

00

03629

213

11012

012552

00

010

110928

9106

00

03723

206

12217

014983

00

020

40476

9296

00

04527

100

11981

01500

9314

00

608064

9951

581

2804

1309

1980

010257

891

14905

2028

1559

6939


（3

（3 （2

OO O

OO O

O

OO

O OO

O

O

OO

NH

NH

HN

HN

HN

HNHNH

HO

HOOH

COOH

COOH

COOHCOOH

NHN

HN

HN

N

N



Data Availability




Acknowledgments



References










































Arthritis










AddictionJournal of







Journal of


Pharmaceutics



Volume 2018



Canadian Journal of






of



0

20

40

60

80

100

120

140

160

k (M

-1s-1

5 6 7 8 9 104pH

)





(ppm)

Temperature∘C


kMSMminus1secminus1











PPIAMSMSELISAMSMS


0

05

1

15

2

25

3

35

4

45

Ratio

to M

SMS








MCLAOP1OP9OP11OP13


100

1000

10000

100000

1000000

log(

Peak

Are

a)






46H69N7O14 [M+H]+ =






46H67N7O12

104 -22

O

O

O

OO

OO

O

NH

NH

NHN

HNNH

NH

COOH

COOH

1 7103356 C31H47N7O12

895 05

O

OO

O

OO

OO

O

N

NH

NH

HN

HN NH

NH

COOH

COOH

2 7123154 C30H45N7O13

na na

O

O

O

OOO

O

N

OO

NH

NH

COOH

COOH

HN

HN

HO

NH

NH6 7423254 C

31H47N7O14

057 -18

O

OH

HO O

O

OOO

OO

O

NH

NH

COOH

COOH

NHN

HNNH

（2

8 7603360 C31H49N7O15

055 335

O

OO OOO

OO O

NH

NH

COOH

COOH

NHN

HN

NH

NH9 9124714 C

45H65N7O13

989 -217

O

OO

OO

OO O

NH

NH

COOH

COOH

NHN

HN

NH

NH

HO（3 11 9424819 C

46H67N7O14

961 173


Table 5 Continued


OO

O

OO

OO

O

NH

NH

COOH

COOH

OH

N

HN

HN

NH

NH

（3 12 9444612 C45H66N7O15

911 321

O

O O

O

OO

OO

NH

NH

COOH

COOHOH

HONHN

HNNH

（3

（2

13 9604925 C46H69N7O15

893 -02

O

O

O

OOO

O

O

O

NH

NH

COOH

COOH

OH

OH

NHN

HNNH

（3

（2

14 9624718 C45H67N7O16

895 98

O

O OO

OO

OO O

NH

NH

N

COOH

COOH

HO

OH

HN

HNHN

NH

（3

15 9744718 C46H67N7O16

890 -037

O

OO

OO

O

O O

O

N

N

HNH

COOH

COOH

OH

HO

HOHN

HN

NH

（3

（2

16 9924823 C46H69N7O17

815 -153




4 Conclusions



Table6Prop

osed

MC-

LAOPanalogou

stothe17MC-

LROPidentifi

edby

Kim

etal[21]Only12

ofthe17prod

uctswe

reidentifi

edin

ourstudies

with

MC-

LA

MCL

AOP1

OP2

OP6

OP8

OP9

OP10

OP11

OP12

OP13

OP14

OP15

OP16

MZ

9104921

7103356

7123149

7423254

760336

9124714

9304819

9424819

9444612

9604925

9624718

9744718

9924823

Time(m)

Area

2189187

1504

00

01750

444

2746

02854

00

04

167826

4368

00

01834

100

7539

07296

00

06

151568

7547

00

03629

213

11012

012552

00

010

110928

9106

00

03723

206

12217

014983

00

020

40476

9296

00

04527

100

11981

01500

9314

00

608064

9951

581

2804

1309

1980

010257

891

14905

2028

1559

6939


（3

（3 （2

OO O

OO O

O

OO

O OO

O

O

OO

NH

NH

HN

HN

HN

HNHNH

HO

HOOH

COOH

COOH

COOHCOOH

NHN

HN

HN

N

N



Data Availability




Acknowledgments



References










































Arthritis










AddictionJournal of







Journal of


Pharmaceutics



Volume 2018



Canadian Journal of






of



PPIAMSMSELISAMSMS


0

05

1

15

2

25

3

35

4

45

Ratio

to M

SMS








MCLAOP1OP9OP11OP13


100

1000

10000

100000

1000000

log(

Peak

Are

a)






46H69N7O14 [M+H]+ =






46H67N7O12

104 -22

O

O

O

OO

OO

O

NH

NH

NHN

HNNH

NH

COOH

COOH

1 7103356 C31H47N7O12

895 05

O

OO

O

OO

OO

O

N

NH

NH

HN

HN NH

NH

COOH

COOH

2 7123154 C30H45N7O13

na na

O

O

O

OOO

O

N

OO

NH

NH

COOH

COOH

HN

HN

HO

NH

NH6 7423254 C

31H47N7O14

057 -18

O

OH

HO O

O

OOO

OO

O

NH

NH

COOH

COOH

NHN

HNNH

（2

8 7603360 C31H49N7O15

055 335

O

OO OOO

OO O

NH

NH

COOH

COOH

NHN

HN

NH

NH9 9124714 C

45H65N7O13

989 -217

O

OO

OO

OO O

NH

NH

COOH

COOH

NHN

HN

NH

NH

HO（3 11 9424819 C

46H67N7O14

961 173


Table 5 Continued


OO

O

OO

OO

O

NH

NH

COOH

COOH

OH

N

HN

HN

NH

NH

（3 12 9444612 C45H66N7O15

911 321

O

O O

O

OO

OO

NH

NH

COOH

COOHOH

HONHN

HNNH

（3

（2

13 9604925 C46H69N7O15

893 -02

O

O

O

OOO

O

O

O

NH

NH

COOH

COOH

OH

OH

NHN

HNNH

（3

（2

14 9624718 C45H67N7O16

895 98

O

O OO

OO

OO O

NH

NH

N

COOH

COOH

HO

OH

HN

HNHN

NH

（3

15 9744718 C46H67N7O16

890 -037

O

OO

OO

O

O O

O

N

N

HNH

COOH

COOH

OH

HO

HOHN

HN

NH

（3

（2

16 9924823 C46H69N7O17

815 -153




4 Conclusions



Table6Prop

osed

MC-

LAOPanalogou

stothe17MC-

LROPidentifi

edby

Kim

etal[21]Only12

ofthe17prod

uctswe

reidentifi

edin

ourstudies

with

MC-

LA

MCL

AOP1

OP2

OP6

OP8

OP9

OP10

OP11

OP12

OP13

OP14

OP15

OP16

MZ

9104921

7103356

7123149

7423254

760336

9124714

9304819

9424819

9444612

9604925

9624718

9744718

9924823

Time(m)

Area

2189187

1504

00

01750

444

2746

02854

00

04

167826

4368

00

01834

100

7539

07296

00

06

151568

7547

00

03629

213

11012

012552

00

010

110928

9106

00

03723

206

12217

014983

00

020

40476

9296

00

04527

100

11981

01500

9314

00

608064

9951

581

2804

1309

1980

010257

891

14905

2028

1559

6939


（3

（3 （2

OO O

OO O

O

OO

O OO

O

O

OO

NH

NH

HN

HN

HN

HNHNH

HO

HOOH

COOH

COOH

COOHCOOH

NHN

HN

HN

N

N



Data Availability




Acknowledgments



References










































Arthritis










AddictionJournal of







Journal of


Pharmaceutics



Volume 2018



Canadian Journal of






of



MCLAOP1OP9OP11OP13


100

1000

10000

100000

1000000

log(

Peak

Are

a)






46H69N7O14 [M+H]+ =






46H67N7O12

104 -22

O

O

O

OO

OO

O

NH

NH

NHN

HNNH

NH

COOH

COOH

1 7103356 C31H47N7O12

895 05

O

OO

O

OO

OO

O

N

NH

NH

HN

HN NH

NH

COOH

COOH

2 7123154 C30H45N7O13

na na

O

O

O

OOO

O

N

OO

NH

NH

COOH

COOH

HN

HN

HO

NH

NH6 7423254 C

31H47N7O14

057 -18

O

OH

HO O

O

OOO

OO

O

NH

NH

COOH

COOH

NHN

HNNH

（2

8 7603360 C31H49N7O15

055 335

O

OO OOO

OO O

NH

NH

COOH

COOH

NHN

HN

NH

NH9 9124714 C

45H65N7O13

989 -217

O

OO

OO

OO O

NH

NH

COOH

COOH

NHN

HN

NH

NH

HO（3 11 9424819 C

46H67N7O14

961 173


Table 5 Continued


OO

O

OO

OO

O

NH

NH

COOH

COOH

OH

N

HN

HN

NH

NH

（3 12 9444612 C45H66N7O15

911 321

O

O O

O

OO

OO

NH

NH

COOH

COOHOH

HONHN

HNNH

（3

（2

13 9604925 C46H69N7O15

893 -02

O

O

O

OOO

O

O

O

NH

NH

COOH

COOH

OH

OH

NHN

HNNH

（3

（2

14 9624718 C45H67N7O16

895 98

O

O OO

OO

OO O

NH

NH

N

COOH

COOH

HO

OH

HN

HNHN

NH

（3

15 9744718 C46H67N7O16

890 -037

O

OO

OO

O

O O

O

N

N

HNH

COOH

COOH

OH

HO

HOHN

HN

NH

（3

（2

16 9924823 C46H69N7O17

815 -153




4 Conclusions



Table6Prop

osed

MC-

LAOPanalogou

stothe17MC-

LROPidentifi

edby

Kim

etal[21]Only12

ofthe17prod

uctswe

reidentifi

edin

ourstudies

with

MC-

LA

MCL

AOP1

OP2

OP6

OP8

OP9

OP10

OP11

OP12

OP13

OP14

OP15

OP16

MZ

9104921

7103356

7123149

7423254

760336

9124714

9304819

9424819

9444612

9604925

9624718

9744718

9924823

Time(m)

Area

2189187

1504

00

01750

444

2746

02854

00

04

167826

4368

00

01834

100

7539

07296

00

06

151568

7547

00

03629

213

11012

012552

00

010

110928

9106

00

03723

206

12217

014983

00

020

40476

9296

00

04527

100

11981

01500

9314

00

608064

9951

581

2804

1309

1980

010257

891

14905

2028

1559

6939


（3

（3 （2

OO O

OO O

O

OO

O OO

O

O

OO

NH

NH

HN

HN

HN

HNHNH

HO

HOOH

COOH

COOH

COOHCOOH

NHN

HN

HN

N

N



Data Availability




Acknowledgments



References










































Arthritis










AddictionJournal of







Journal of


Pharmaceutics



Volume 2018



Canadian Journal of






of





46H67N7O12

104 -22

O

O

O

OO

OO

O

NH

NH

NHN

HNNH

NH

COOH

COOH

1 7103356 C31H47N7O12

895 05

O

OO

O

OO

OO

O

N

NH

NH

HN

HN NH

NH

COOH

COOH

2 7123154 C30H45N7O13

na na

O

O

O

OOO

O

N

OO

NH

NH

COOH

COOH

HN

HN

HO

NH

NH6 7423254 C

31H47N7O14

057 -18

O

OH

HO O

O

OOO

OO

O

NH

NH

COOH

COOH

NHN

HNNH

（2

8 7603360 C31H49N7O15

055 335

O

OO OOO

OO O

NH

NH

COOH

COOH

NHN

HN

NH

NH9 9124714 C

45H65N7O13

989 -217

O

OO

OO

OO O

NH

NH

COOH

COOH

NHN

HN

NH

NH

HO（3 11 9424819 C

46H67N7O14

961 173


Table 5 Continued


OO

O

OO

OO

O

NH

NH

COOH

COOH

OH

N

HN

HN

NH

NH

（3 12 9444612 C45H66N7O15

911 321

O

O O

O

OO

OO

NH

NH

COOH

COOHOH

HONHN

HNNH

（3

（2

13 9604925 C46H69N7O15

893 -02

O

O

O

OOO

O

O

O

NH

NH

COOH

COOH

OH

OH

NHN

HNNH

（3

（2

14 9624718 C45H67N7O16

895 98

O

O OO

OO

OO O

NH

NH

N

COOH

COOH

HO

OH

HN

HNHN

NH

（3

15 9744718 C46H67N7O16

890 -037

O

OO

OO

O

O O

O

N

N

HNH

COOH

COOH

OH

HO

HOHN

HN

NH

（3

（2

16 9924823 C46H69N7O17

815 -153




4 Conclusions



Table6Prop

osed

MC-

LAOPanalogou

stothe17MC-

LROPidentifi

edby

Kim

etal[21]Only12

ofthe17prod

uctswe

reidentifi

edin

ourstudies

with

MC-

LA

MCL

AOP1

OP2

OP6

OP8

OP9

OP10

OP11

OP12

OP13

OP14

OP15

OP16

MZ

9104921

7103356

7123149

7423254

760336

9124714

9304819

9424819

9444612

9604925

9624718

9744718

9924823

Time(m)

Area

2189187

1504

00

01750

444

2746

02854

00

04

167826

4368

00

01834

100

7539

07296

00

06

151568

7547

00

03629

213

11012

012552

00

010

110928

9106

00

03723

206

12217

014983

00

020

40476

9296

00

04527

100

11981

01500

9314

00

608064

9951

581

2804

1309

1980

010257

891

14905

2028

1559

6939


（3

（3 （2

OO O

OO O

O

OO

O OO

O

O

OO

NH

NH

HN

HN

HN

HNHNH

HO

HOOH

COOH

COOH

COOHCOOH

NHN

HN

HN

N

N



Data Availability




Acknowledgments



References










































Arthritis










AddictionJournal of







Journal of


Pharmaceutics



Volume 2018



Canadian Journal of






of



Table 5 Continued


OO

O

OO

OO

O

NH

NH

COOH

COOH

OH

N

HN

HN

NH

NH

（3 12 9444612 C45H66N7O15

911 321

O

O O

O

OO

OO

NH

NH

COOH

COOHOH

HONHN

HNNH

（3

（2

13 9604925 C46H69N7O15

893 -02

O

O

O

OOO

O

O

O

NH

NH

COOH

COOH

OH

OH

NHN

HNNH

（3

（2

14 9624718 C45H67N7O16

895 98

O

O OO

OO

OO O

NH

NH

N

COOH

COOH

HO

OH

HN

HNHN

NH

（3

15 9744718 C46H67N7O16

890 -037

O

OO

OO

O

O O

O

N

N

HNH

COOH

COOH

OH

HO

HOHN

HN

NH

（3

（2

16 9924823 C46H69N7O17

815 -153




4 Conclusions



Table6Prop

osed

MC-

LAOPanalogou

stothe17MC-

LROPidentifi

edby

Kim

etal[21]Only12

ofthe17prod

uctswe

reidentifi

edin

ourstudies

with

MC-

LA

MCL

AOP1

OP2

OP6

OP8

OP9

OP10

OP11

OP12

OP13

OP14

OP15

OP16

MZ

9104921

7103356

7123149

7423254

760336

9124714

9304819

9424819

9444612

9604925

9624718

9744718

9924823

Time(m)

Area

2189187

1504

00

01750

444

2746

02854

00

04

167826

4368

00

01834

100

7539

07296

00

06

151568

7547

00

03629

213

11012

012552

00

010

110928

9106

00

03723

206

12217

014983

00

020

40476

9296

00

04527

100

11981

01500

9314

00

608064

9951

581

2804

1309

1980

010257

891

14905

2028

1559

6939


（3

（3 （2

OO O

OO O

O

OO

O OO

O

O

OO

NH

NH

HN

HN

HN

HNHNH

HO

HOOH

COOH

COOH

COOHCOOH

NHN

HN

HN

N

N



Data Availability




Acknowledgments



References










































Arthritis










AddictionJournal of







Journal of


Pharmaceutics



Volume 2018



Canadian Journal of






of



Table6Prop

osed

MC-

LAOPanalogou

stothe17MC-

LROPidentifi

edby

Kim

etal[21]Only12

ofthe17prod

uctswe

reidentifi

edin

ourstudies

with

MC-

LA

MCL

AOP1

OP2

OP6

OP8

OP9

OP10

OP11

OP12

OP13

OP14

OP15

OP16

MZ

9104921

7103356

7123149

7423254

760336

9124714

9304819

9424819

9444612

9604925

9624718

9744718

9924823

Time(m)

Area

2189187

1504

00

01750

444

2746

02854

00

04

167826

4368

00

01834

100

7539

07296

00

06

151568

7547

00

03629

213

11012

012552

00

010

110928

9106

00

03723

206

12217

014983

00

020

40476

9296

00

04527

100

11981

01500

9314

00

608064

9951

581

2804

1309

1980

010257

891

14905

2028

1559

6939


（3

（3 （2

OO O

OO O

O

OO

O OO

O

O

OO

NH

NH

HN

HN

HN

HNHNH

HO

HOOH

COOH

COOH

COOHCOOH

NHN

HN

HN

N

N



Data Availability




Acknowledgments



References










































Arthritis










AddictionJournal of







Journal of


Pharmaceutics



Volume 2018



Canadian Journal of






of



（3

（3 （2

OO O

OO O

O

OO

O OO

O

O

OO

NH

NH

HN

HN

HN

HNHNH

HO

HOOH

COOH

COOH

COOHCOOH

NHN

HN

HN

N

N



Data Availability




Acknowledgments



References










































Arthritis










AddictionJournal of







Journal of


Pharmaceutics



Volume 2018



Canadian Journal of






of
































Arthritis










AddictionJournal of







Journal of


Pharmaceutics



Volume 2018



Canadian Journal of






of


permanganate oxidation of microcystin-la: kinetics...

Documents