Identification of Unknown Microcystins in Alberta Lake Water

Identification of Unknown Microcystins in Alberta Lake Water Application Note Environmental Authors Ralph Hindle Vogon Laboratory Services Ltd. ochrane, Alberta anada Xu Zhang and David Kinniburgh Alberta entre for Toxicology Alberta Health & Wellness University of algary algary, Alberta anada Abstract Detection, characterization, and tentative identification of very low levels of unknown microcystins in lake water are possible in the absence of analytical standards using a combination of triple quadrupole L/MS and L/Q-TF analysis and a Personal ompound Database (PD) compiled from the Word Health rganization (WH) list of microcystins.

Introduction The occurrence of cyanobacterial toxins in anadian fresh waters is a serious environmental and public health concern [1]. Microcystins (Ms) are a class of common cyanobacterial toxins present in anadian lakes, and they are potent inhibitors of eukaryotic protein phosphatases []. Microcystins are also powerful hepatotoxins, and may promote tumor development in mammals, presenting a serious threat to livestock and human drinking water sources. These cyanobacterial toxins have been detected in every province in anada, often at levels above maximum guidelines for recreational water quality []. The Ms are cyclic peptides containing seven amino acids. They have the general structure shown in Figure 1. Table 1 gives the chemical structures for eight microcystins surveyed in Alberta lakes. The most frequently reported M is LR, containing leucine and arginine at positions R and R, respectively. The Alberta entre for Toxicology (AFT) conducted a comprehensive study of microcystins in Alberta lakes, and it continues to monitor fresh water sources using liquid chromatography and triple quadrupole mass spectrometry. While this method tests for eight microcystins, a nontargeted compound was also detected in a sample with the same transition qualifier ion as M YR (m/z 1), but it had an incorrect retention time (RT) and qualifier ion ratio. This application note describes two methods developed through a collaboration between Alberta entre for Toxicology (AFT) and Vogon Laboratory Services to provide sensitive detection of a wide range of microcystins and establish the identity of this newly observed compound. A confirmation method was first developed using an Agilent 19 Infinity L System and an Agilent Triple Quadrupole L/MS, with a different retention time pattern from the reference AFT method. Accurate mass determination on an Agilent Q-TF L/MS system was then used to provide tentative identification of the unknown peak as desmethylated microcystin HtyR. H 3 Glu H Figure 1. General microcystin (M) structure. Mdha 7 N Adda HN Ala 1 H H 3 R R General microcystin structure Position Abbreviation Amino acid R 1 Ala 1 Alanine R Leu (L) Leucine R 3 MeAsp 3 Methylaspartic acid R Arg (R) Arginine MeAsp 3 H R Adda 3-amino-9-methoxy-,,-trimethyl- 1-phenyldeca-(E),(E)-dienoic acid R Glu Glutamic acid R 7 Mdha 7 N-methyldehydroalanine Table 1. Eight Microcystins Surveyed in Alberta Lakes* Microcystin R R Formula Neutral mass LR Leucine Arginine 9 H 7 N 1 1 99.88 Desmethyl LR Leucine Arginine 8 H 7 N 1 1 98.331 RR Arginine Arginine 9 H 7 N 13 1 137.8 YR Tyrosine Arginine H 7 N 1 13 1.8 LA Leucine Alanine H 7 N 7 1 99.88 LW Leucine Phenylalanine H 7 N 8 1 1.7 LF Leucine Tryptophan H 71 N 7 1 98.11 HtyR Homotyrosine Arginine 3 H 7 N 1 13 18.37 *See Figure 1 for the general microcystin structure All microcystins were detected at.1 ng/ml, which is well below the 7 anadian Drinking Water Guideline of 1. µg/l. Using a Personal ompound Database (PD) of compounds created in the Agilent MassHunter software suite from the World Health rganization (WH) list of microcystins, the Q-TF data enabled the tentative identification of an additional seven microcystins.

Experimental Instruments The confirmation method was developed on an Agilent 19 Infinity L System equipped with an Agilent GA Autosampler and coupled to an Agilent Triple Quadrupole L/MS System. The instrument conditions are listed in Table. The method for tentative identification of unknown microcystins was developed on a 19 Infinity L system equipped with a GA Autosampler and coupled to an Agilent B Q-TF L/MS System with Jet Stream electrospray source. The instrument conditions are listed in Table. Table. L and Triple Quadrupole MS Run onditions L conditions olumn Agilent Poroshell SB-18, 3. 1 mm,.7 µm (p/n 897-3) olumn temperature Injection volume µl Mobile phase A) 1 mm ammonium fluoride in water (HPL grade) B) % isopropanol in acetonitrile (L/MS grade) Autosampler temperature Flow rate. ml/min Gradient Time (min) % B 3. 3.. 1 Stop 7 minutes Post time minutes Triple quadrupole MS conditions Ionization mode ESI with Agilent Jet Stream Technology Drying gas temperature 3 Drying gas flow 1 L/min Nebulizer pressure psig Sheath gas temperature Sheath gas flow 11 L/min apillary voltage, V Nozzle voltage 1, V EMV V Q-TF MS conditions Mode Targeted MS/MS Acquisition Profile and centroid; GHz Range 1 1,7 amu Acquisition rate (MS) 3 scans/s Acquisition rate (MS/MS) 1 scan/s Reference masses 11.9 and 9.98 Analysis parameters The Triple Quadrupole L/MS multiple reaction monitoring (MRM) analysis parameters are shown in Table 3. Table 3. Microcystin MRM Analysis Parameters a for the Target ompounds Using a Triple Quadrupole L/MS Precursor b (m/z) LR 99. Desmethyl-LR 981. RR. YR 1. LA 91. LY 1. LW 1. LF 98. HtyR 19. Product ion (m/z) 13. 8 13. 8 13. 8 13. 8 13. 3 13. 13. 8 13. 7 13. 7 13. 7 13. 8 13. 7 13. 8 13. 13. 7 13. 13. 8 13. 7 ollision energy (V) a The fragmentor and cell acceleration voltages were 1 V and V, respectively, for all transitions. b All precursors are singly charged, except RR which is doubly charged. Sample preparation Water samples (1 ml) were taken through three freeze/thaw cycles, then sonicated for minutes, followed by filtration through.-µm cellulose filters directly into autosampler vials. 3

Results and Discussion Identifying an unknown microcystin ne sample analyzed using the AFT method revealed a compound with the same transition and qualifier ion as YR (m/z 1), but at the wrong retention time (RT) and qualifier ion ratio. It was tentatively identified as desmethyl-htyr, based on the loss of 1 amu from HtyR (m/z 19). To confirm the identification of this unknown M, a triple quadrupole L/MS confirmation method was developed, which provided a different retention time pattern from the reference AFT method. Additional microcystin analytes were added to the method to help characterize compounds. Finally, samples were also analyzed using an L/Q-TF method to confirm the identity of the unknown compound. onfirmation method The triple quadrupole L/MS confirmation method was optimized to provide a minimum detection level at approximately 1% of the anadian Drinking Water Guidelines (DWG), which is 1. µg/l, based on M LR. The lowest level calibrators used for the method were.1 µg/l for Ms YR, LR, and RR, and. µg/l for all others. Figure illustrates the complete separation achieved for nine Ms with the high performance liquid chromatography (HPL) chromatographic method at both the lowest calibrator levels and calibrator levels near the DWG. 1 1.. 1 1 1 1.. 1 1 1.. 1 1 1 1 1 1.. 1 1 A.8.38.7.....8 Lowest level calibrator.1 µg/l for YR, LR, and RR. µg/l for all others.8.97.98 3..37.1 3. 3. 3. 3. 3.8.... Acquisition time (min) 1 B. YR.. 1 HtyR.33 1 3 1. LR.1. 1 dm-lr.78 1 LA.9 1 3.97 RR 1 LY 3. 1 LW.3 1. LF.......8 3. 3. 3. 3. 3.8.... Acquisition time (min) alibrator near DW guidelines.9 µg/l for YR, LR, and RR 1.9 µg/l for all others Figure. Example total ions chromatograms (TIs) for nine microcystins at concentrations approximately 1% (A) and 1% (B) of the DWG of 7.

Quantitation calibration coefficients (R ) were excellent, ranging from.9978 to.999 for the six Ms using a minimum calibrator level of. µg/l and a maximum calibrator level of µg/l (Table ). The R values ranged from.999 to.9997 for the three Ms using a minimum calibrator level of.1 µg/l and a maximum calibrator level of µg/l. A representative calibration curve for M LR is shown in Figure 3, illustrating excellent linearity even at extremely low concentrations. Accuracy ranged from 8% to 11% for the six Ms with a minimum calibrator level of. µg/l, and 11% to 119% for the three Ms with a minimum calibrator level of.1 µg/l (Table ). Analyzing the unknown Using the confirmation method to analyze the unknown M from an Alberta lake water sample showed a peak with the same transition and qualifier ions as YR, but with an RT of 1.91 minutes, rather than. minutes. The qualifier ion ratio (m/z 13./13.) was also too low to be YR (% versus %). The low abundance of the m/z 13. peak indicates possible desmethylation of the N-methyldehydroalanine (Mdha) moiety in the YR structure. Unfortunately, desmethyl HtyR is not commercially available. Therefore, Q-TF for accurate mass analysis was used to further confirm the desmethylation hypothesis. Table. alibration oefficients and Accuracy of Recovery alibration range M R Accuracy a (%) Hty R.999 9 Desmethyl-LR.9993 8. to µg/l LA.9988 11 LY.9998 9 LW.9989 118 LF.9978 119 YR.9993 118.1 to µg/l LR.9997 11 RR.999 119 a Quantitative accuracy is listed for calibrator 1 (.1 or. µg/l) Responses 1 1. 1.8... 8 1 1 1 1 18 oncentration (ng/ml) 1 3 3. 3. 1. 1. Responses Expanded range from.1 to.9 µg/l.1..3....7.8.9 1 oncentration (ng/ml) Figure 3. A calibration curve for M LR using three-fold serial dilutions from.1 to µg/l, including the expanded range from.1 to.9 µg/l, to illustrate excellent linearity.

L/Q-TF analysis The accurate mass determined for the precursor ion for the unknown M using liquid chromatography/q-tf MS (L/Q-TF) was 19., which matches the actual mass for desmethylated HtyR with a mass error of. ppm. This further supports the hypothesis that the unknown is formed by the desmethylation of HtyR. Examination of the transition ions reveals that both the unknown and YR contain the two m/z 13 nominal mass ions characteristic for microcystins (Figure ). The actual masses of the two ions are 13.8 and 13.118, formed by different fragmentation patterns of the Adda group. Both ions are well separated in both microcystins at mass resolution 1,. 1 3 1. 1. 1..8... 1 3 1. 1. 1. 1..8... 13.798 1,79 13.79 13,998 13.118 1,383 13.117 1,37 YR 13.71 11, Unknown microcystin 13.1188 13,8 13.81 13.118 1,9 137.783 1,37 13.8 13 13. 13. 13. 13.8 13 13. 13. 13. 13.8 137 137. 137. Mass-to-charge (m/z) 13.8 = 9 H 11 + 13.118 = 1 H 1 + H 3 H 3 H HN H HN Z N H H 3 H N H H 3 H The two Adda ions formed from all microcystins during analysis MS. Figure. The two ions derived from the Adda group and characteristic of all microcystins are present and well separated in both YR and the unknown microcystin, using L/Q-TF. Z X X

However, the m/z 13 ion formed from cleavage of the Glu + Mdha groups from the microcystins is present as expected in M YR, but present at only trace levels in the unknown M (Figure ). This indicates that the unknown M is not YR. Desmethylation of the Glu + Mdha group would be expected to produce an ion with an accurate mass of 199.713 (Figure ). This ion is observed in the spectra from the unknown M, but not in the spectra of M YR. These results also support the hypothesis that the unknown M is dm-htyr. A previous structural L/MS/MS characterization of microcystins had identified eight major ions designated a-h [3]. Two of these, ions f and h, can be used to confirm the identity of the unknown M. Ion f contains the R 7 and R moieties (Figure 1), which should be dm-mdha (Dha) and Hty, respectively for the microcystin dm-htyr. Ion h contains the R moiety, as well as a second possible desmethylation site, MeAsp 3. Depending on the identities of R 7 and R and the presence or absence of desmethylation at position R 3, the accurate masses of these two ions will vary and be diagnostic of the microcystin structure. 1 3 1 1 3 1 YR Unknown microcystin 13.8 13.13 1.899 13.13 1 1. 1. 1. 1.8 13. 13. 13. 13. 13.8 1 1. 1. Mass-to-charge (m/z) H 3 Glu + Mdha 7 = 9 H 13 N + m/z = 13.87 H HN R N H H 3 H Figure. Q-TF spectra of YR and the unknown microcystin in the m/z range from 1 to 1.. The m/z 13 ion characteristic of the Glu + Mdha group is present in microcystin YR but at low abundance in the unknown M. R 1.. YR.113 1. 1.. 1.. 1. 1. Unknown microcystin 199.9.119 1.98 Desmethylation of Glu + Mdha 8 H 11 N + fragment ion from [Glu + Mdha 7 H ] will have accurate mass 199.713 1.99. 198.11 198 198. 199 199.. 1 1.. Mass-to-charge (m/z) Figure. Q-TF spectra of YR and the unknown microcystin in the m/z range from 198 to. The m/z 199 peak is present in the unknown microcystin, indicating desmethylation of the Glu + Mdha group. This peak is not present in YR. 7

Analysis by Q-TF of ion f in the unknown microcystin revealed accurate masses that confirmed the presence of Dha at position 7 and Hty at position, consistent with the identification of the unknown as dm-htyr. Analysis of the HtyR standard also gave the correct accurate mass for ion f (Figure 7). 1 3 1..9.8.7....3..1 1... 1.7 1. 1. 1..7.. HtyR Unknown microcystin 3.88 198 198. 199 199. Mass-to-charge (m/z) 17.39.313. 1 Sequence for Ion f: R 7 _ Ala 1_ R _ MeAsp 3_ Arg Structure of M HtyR: R 7 = Mdha; R = Hty alc m/z: 17.3 Measured: 17.39 (.8 ppm mass error) Proposed structure (dm-htyr) for the unknown compound, with desmethylation of Mdha 7 to Dha 7 : R 7 = Dha; R = Hty alc m/z: 3.88 Measured: 3.88 (. ppm mass error) Figure 7. Q-TF spectra of ion f for HtyR and the unknown microcystin. The accurate mass for the f ion observed in HtyR (upper) matched the calculated mass for the ion containing Hty at position 7 and Mdha at position R 3. In contrast, the mass observed for ion f in the unknown M matched the calculated mass for the ion containing Hty at position 7 and Dha at position R 3. 8

The spectra of the h ion confirmed that minimal desmethylation is occurring in the R 3 position in the unknown M, as its accurate mass matched the calculated mass for methylated aspartic acid in position 3, in both the unknown and the HtyR standard (Figure 8). 1. 3.9 Sequence for Ion h: Ala 1 _ R _ MeAsp 3_ Arg 3. 3... Structure of M HtyR: R = Hty alc m/z: 3.71 Measured: 3.9 (.3 ppm mass error) 1. 1....3 3.87 1 1 3.83 Proposed structure (dm-htyr) for the unknown compound, with no desmethylation at position 3: 3 R 7 = Hty alc m/z: 3.71 Measured: 3.83 (. ppm mass error) 1.38.9 9.78 8 3 3 3 3 Mass-to-charge (m/z) Figure 8. Q-TF spectra of ion h for HtyR and the unknown microcystin. The accurate mass for the h ion observed in both HtyR (upper) and the unknown M matched the calculated mass for the ion containing Hty at position R 7 and Mdha at position R 3. In contrast, only a very small peak was observed at nominal mass m/z in both microcystins, indicating that little or no desmethylation was occurring at position R 3. 9

Using a Personal ompound Database to identify multiple unknown microcystins The accurate mass capabilities of the Q-TF can be used to tentatively identify other microcystins that may be present in Alberta lakes, based only on the mass of their H+ adducts. The first step in the process is to build a Personal ompound Database (PD) using the Agilent MassHunter PDL Manager Software, which enables the user to create and edit a customizable PD, including compounds, accurate-mass, and retention time information. ne of the advantages of accurate mass scan data is the ability to retrospectively search acquired data for new compounds using such a database. In this study, the WH list of microcystins [] was used to enter the formulas for microcystins into a PD, which generates accurate masses for the H+ adducts. The Find by Formula tool in MassHunter was then used to search for these accurate masses in the sample total ions chromatogram (TI) data file. Using this process with the Q-TF, seven unknowns were tentatively identified in samples from Lake A and Lake B, in addition to the desmethyl HtyR (Figures 9 and 1). Additional analysis by Q-TF MS/MS and comparison to analytical standards is needed to confirm the presence and identity of these Ms. Work is on-going to develop an exactmass calculator model to help identify nontargeted Ms and their variants. 1 7. 7....... 3. 3... 1. 1.. Lake A..17. 38,13,1.9,731. 1,83,11.9 3,788.7,7.8 3,97.97 8,.1 3,3.1..3....7.8.9..1..3 Acquisition time (min) RT ompound name Formula m/z Area Score Mass diff (ppm)*. M-YR H7 N1 13 1.3 1,83,11 98. -.7.9 M-LR 9 H7 N1 1 99.9 3,788 9. -.1.7 M-HtyR-DAsp3-Dha7 1 H7 N1 13 131.18,7 9.8 -.7.7 M-HtyR 3 H7 N1 13 19.1 9,97 98. -.3.8 M-DesMe-LR 8 H7 N1 1 981. 3,97 99.1 -.1.97 M-HphR-Dha7 H7 N1 1 19. 8, 99. -..99 M-LR-DAsp3-Dha7 7 H7 N1 1 97.3 11,8 9.3-1..99 M-LR 9 H7 N1 1 99.9 3,18 9.7 -. *Mass difference determined by subtracting the theoretical mass of the compound from the calculated mass derived from the Q-TF analysis, expressed in ppm. Figure 9. Extracted ion chromatograms of a water sample from Lake A, and a table including retention time (RT), compound name, calculated m/z, and difference of the calculated mass from the theoretical mass for each unknown tentatively identified using the Find by Formula tool in MassHunter and the personal compound database (PD) created for the microcystins in the WH list. A mass tolerance of ppm and a minimum match score of 7 were used to make the identification of H+ adducts. 1

1 1. 1. 1. 1. Lake B. 3,8,7.81 3,7,9.8.....13 1,,31.3 8,. 17,1.93,3. 38,79.1..3....7.8.9..1..3. Acquisition time (min) RT ompound name Formula m/z Area Score Mass diff (ppm)*.13 M-HtyR-DAsp3-Dha7 1 H7 N1 13 131.193 1,,31 99. -..3 M-LR-DAsp3-Dha7 7 H7 N1 1 97. 8, 99.7 -.3. M-LR-DAsp3-Dha7 7 H7 N1 1 97. 17,1 8..7. M-HtyR-DAsp3-ADMAdda-Dhb7 3 H7 N1 1 173.3 3,8,7 99...8 M-LR 9 H7 N1 1 99.9 18,83 97.7-1.1.81 M-LR-DAsp3-ADMAdda-Dhb7 9 H7 N1 13 19.39 3,7,9 99...93 M-LY H71 N7 13 1.181,3 99. -.3. M-LR-ADMAdda H7 N1 13 13. 38,79 9.9 -. *Mass difference determined by subtracting the theoretical mass of the compound from the calculated mass derived from the Q-TF analysis, expressed in ppm. Figure 1. Extracted ion chromatograms of a water sample from Lake B, and a table including retention time (RT), compound name, calculated m/z, and difference of the calculated mass from the theoretical mass for each unknown tentatively identified using the Find by Formula tool in MassHunter and the personal compound database (PD) created for the microcystins in the WH list. A mass tolerance of ppm and a minimum match score of 7 were used to make the identification of H+ adducts. onclusion When analytical standards are available, the Agilent 19 Infinity L System and an Agilent Triple Quadrupole L/MS provides an excellent platform for the targeted analysis of microcystins. Analysis by triple quadrupole L/MS using the Agilent Q-TF L/MS System can confirm suspect compounds using the accurate mass of the molecular ion adducts, as well as MS/MS fragments. The combination of these two technologies supported the hypothesis that dm-htyr was present in an Alberta lake water sample. Databases can be compiled using the Agilent MassHunter PDL Manager Software to include the chemical formula for additional microcystins based on reported analogues in the literature. Using MassHunter Find by Formula, previously acquired data files can be retrospectively searched against PDL databases and libraries for these additional compounds as they become known. 11

References 1. B.G. Kotak, A.K.-Y. Lam, E. E. Prepas, and S.E. Hrudey. Role of chemical and physical variables in regulating microcystin-lr concentration in phytoplankton of eutrophic lakes. an. J. Fish. Aquat. Sci. 7, 18 193 ().. D. M. rihel, D. F. Bird, M. Brylinsky, H. hen, D. B. Donald, D. Y. Huang, A. Giani, D. Kinniburgh, H. Kling, B. G. Kotak, P. R. Leavitt,.. Nielsen, S. Reedyk, R.. Rooney, S. B. Watson, R. W. Zurawell, and R. D. Vinebrooke. High microcystin concentrations occur only at low nitrogen-tophosphorus ratios in nutrient-rich anadian lakes. an. J. Fish. Aquat. Sci. 9, 17-1 (1). 3. T. Mayumi, H. Kato, S. Imanishi, Y. Kawasaki, M. Hasegawa, K. Harada. Structural characterization of microcystins by L/MS/MS under ion trap conditions. J. Antibiot (Tokyo) 9, 71-719 ().. I. horus and J. Bartram (Eds.). Toxic yanobacteria in Water A Guide to Their Public Health onsequences, Monitoring, and Management. E & FN Spon, published on behalf of the World Health rganization, New York (1999). For More Informatiom For more information on our products and services visit our Website at www.agilent.com/chem. www.agilent.com/chem Agilent shall not be liable for errors contained herein or for incidental or consequential damages in connection with the furnishing, performance, or use of this material. Information, descriptions, and specifications in this publication are subject to change without notice. Agilent Technologies, Inc., 1 Published in the USA April 8, 1 991-EN