Abstract

Abstract

This application note describes the quantitation of 8 individual microcystin (MC) isoforms and Nodularin-R using the SCIEX QTRAP® 4500 System in positive mode electrospray ionization (ESI). Using an 11 min chromatographic separation, excellent sensitivity, accuracy and precision was shown. The calculated lowest concentration minimal reporting levels (LCMRL) for the standards ranged from 4.8 ng/L for MC-RR to 91.8 ng/L for MC-YR, suggesting that the direct analysis of ambient water samples is possible. In the EPA Method 544, a 500-fold concentration factor is advised which equates to LCMRL values of 0.010 to 0.184 ng/L in the water sample for this method.

Introduction

Introduction

Microcystins (MC) and nodularins (NOD) are toxins produced by cyanobacteria in saline and freshwaters. MC and NOD are released during cell death and are potential drinking water contaminants. Therefore, accurate and sensitive methods for quantifying MC and NOD in water samples are needed.

MC and NOD both share the common amino acid ADDA, but MC are cyclic heptapeptides whereas NOD are cyclic pentapeptides. Over 130 MC and 10 NOD isoforms have been identified primarily based on variations of two L-amino acids in their cyclic peptide structure.^1,2

MC and NOD are primarily liver toxicants and toxicity varies by isoform with the Microcystin-LR (leucine/arginine variant) thought to be the most harmful. Therefore, the quantification of individual isoforms in necessary. MC and NOD contamination from harmful algal blooms is widespread in surface and drinking water, resulting in occasional consumption advisories.^3,4 The US EPA 10-day drinking water health advisory for microcystins is 0.3 μg/L for infants and children up to 6 years old, and 1.6 μg/L for adults.⁵ In addition, Health Canada has set a maximum acceptable concentration (MAC) of MC-LR of 1.5 μg/L⁶ and the World Health Organization (WHO) MC-LR provisional guideline is 1 μg/L.⁷ Drinking water guidelines for NOD do not exist.

Figure 1. Low level quantitation of MC-LR. MRM chromatogram for the LOQ standard (43.8 ng/mL) of MC-LR.1 Gaussian smooth performed.

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Methods

Key features of the QTRAP 4500 System for quantitation of MC and NOD

• Targeted LC-MRM method has been developedon the QTRAP 4500 System for the quantitation of microcystinsand nodularins
• Good sensitivity was achieved with Lowest Concentration Minimum Report Level (LCMRL) concentrationsin the ng/L range(Figure 1)
• Fast chromatographic method with baseline separation was developed (11 mins, Figure 2) that was significantly shorter than the established EPA Method 544, providing improved throughput

Methods

Sample preparation: Neat standards were obtained from Enzo Life Sciences (Farmingdale, NY) and reconstituted in 1 ml of methanol. An intermediate mixed stock was prepared by diluting the standards in methanol to yield 500 ng/mL for MC-RR and Nodularin-R, and 2000 ng/ml for MC-LA, MC-LF, MC-LR, MC-LY, MC-LW, MC-YR, MC-WR. Calibration standards were prepared with 5% acetonitrile in water to match the initial LC conditions. Standards were prepared in glass vials to reduce sorption to plastic surfaces. All standards were kept at -20 ºC until analysis. Chromatography: A

SCIEX ExionLC™AC System

was used as the LC system. Chromatographic separation was achieved under gradient conditions using a Phenomenex Kinetex®C8 column (2.6 μm particle size, 100 x 2.1 mm) and flow rate of 0.500 mL/min (Table 1). The mobile phases were water (“A”) and acetonitrile (“B”), both modified with 0.1% formic acid. The column oven was set to 40 ºC and injection volume was 20 μL. To reduce sample carryover the autosampler rinse solvent was 60:20:20 isopropyl alcohol: methanol: acetonitrile using a rinse volume of 2 mL and dip time of 8 sec.

Table 1. LC gradient program.

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Mass Spectrometry: Analysis was performed on a SCIEX QTRAP®4500 System with a Turbo V™ Source using an electrospray ionization (ESI) probe in positive mode. Compound-specific and ion source parameters were manually optimized (Tables 2 & 3) and two MRMs per compound were monitored except for MC-LY which showed only 1 product ion. The Scheduled MRM™ Algorithm was used to maximize dwell times and optimize the number of points across the chromatographic peaks. The MRM detection window was set to 45 s and target scan time was 0.25 s.

Table 2. Ion source parameters

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determination

Data Processing: The standard batch was run 7 times to generate method performance statistics (i.e. accuracy and precision of LOQ standard)as well as to calculate the LCMRL values. Quantification was performed with MultiQuant™ Software 3.0.2 using 1.0 Gaussian smoothing and 1/x or 1/x2 weighted linear regression. The signal/noise ratio was calculated using the peak-to-peak S/N algorithm in PeakView® Software 2.2 on unsmoothed chromatograms. The LOD was determined as S/N>3. The LOQ was determined using the following criteria: S/N>8, at least 8 points across the peak and accuracy between 80-120%. LOQ and LOD concentrations were calculated usingthe first MRM transition, per compound, described in Table 3.

The lowest concentration minimum reporting level (LCMRL) was calculated as described by Winslow et al.8using Excel 2016. The LCMRL values were calculated using the LOD standard and subsequent three standard levels. Briefly, the measured versus actual concentrations were plotted and linear regression calculated. The 99% prediction intervals and data quality objective bounds (50% and 150% sample recovery) were calculated and plotted on the original graph. The LCMRL was defined as the intersection of the upper and lower prediction interval lines with the data quality objective (DQO) bounds, using the higher calculated concentration.

Determination of detection limits

Using the developed gradient program, baseline separation was achieved for all compounds with excellent peak shape (Figure 2). The gradient is 15 min shorter than the program described in EPA Method 544, resulting in considerable time savings but still maintaining baseline separation. The LOD concentrations varied by compound and ranged from 2.7 to 21.9 ng/L (Table 4). Specifically, MC-LA, MC-RR and Nodularin showed the lowest LOD values, whereas MC-LR and MC-YR showed the highest. The LOQ concentrations also varied by compound (5.5-43.8 ng/L) and showed similar trends as the LOD values. MRM chromatograms for the LOQ standard (43.8 ng/L) of MC-LR, following 1 Gaussian smooth, are shown in Figure 1. The reported LOQ concentrations are significantly below the US EPA drinking water advisory level for children of 300 ng/L.

The LOQ standard showed excellent accuracy, with the mean accuracy ranging from 98.1% to 113% (n=7). Further, the precision of the LOQ standard was very good and was generally <20% (n=7). Finally, the LOQ standard signal-to-noise ratio was >10 for all compounds.

The method showed approximately 3 orders of linear dynamic range for all compounds with linearity maintained up to 25,000 ng/L for MC-RR and Nodularin-R, and up to 100,000 ng/L for MC-LA, MC-LF, MC-LW and MC-LY. Previous analysis showed that MC-LR and MC-YR were linear up to 40,000 ng/L.

Figure 2. Extracted ion chromatograms. Overlaid XICs of 21.9 ng/L standard for MC-RR and Nodularin-R, 87.5 ng/L standard for MC-YR, MC-LR, MC-WR, MC-LA, MC-LY, MC-LW and MC-LF using the QTRAP 4500 system. 1 Gaussian smooth performed

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Table 3. MS method information. MRM masses and compound-specific MS parameters for QTRAP 4500 System.

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Table 4. Method performance parameters. Method performance parameters such as sensitivity, linear range, LOQ accuracy and precision, signal-to-noiseare computed for the method. Peak-to-peak S/N was calculated using PeakViewSoftware2.2 with unsmoothed chromatograms.

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computation

Computation of LCMRL values

LCMRL values were calculated using the results of the standards analysis (Table 5). For all compounds, the LCMRL graphs met the required criteria of seven replicate samplesat four concentration levels, and at least one standard level below the calculated LCMRL.⁸ An example LCMRL graph is shown in Figure 3 for MC-LR. The LCMRL values –calculated as “in vial” concentrations –ranged from 4.8 ng/L for MC-RR to 91.8 ng/L forMC-YR. However, EPA Method 544 uses solid phase extraction techniques to clean and concentrate the water samples with a suggested concentration factor of 500-fold. Therefore, the LCMRL values –calculated on “sample” basis –range from 0.10 ng/L to 0.184 ng/mL

Table 5. Lowest concentration minimum report level (LCMRL) concentrations.

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Figure 3. LCMRL graph for MC-LR.

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conclusions

Conclusions

Here, a method for the quantitation of 8 individual microcystin (MC) isoforms and Nodularin-R using the QTRAP 4500 System has been described. A fast 11 minute separation was achieved, maintaining baseline separation of the compounds. Excellent sensitivity, accuracy and precision was shown with lowest concentration minimalreporting levels (LCMRL) for the standards ranged from 4.8 ng/L for MC-RR to 91.8 ng/L for MC-YR. However, EPA Method 544 advises a 500-fold concentration factor which equates to LCMRL values for this method of 0.010 to 0.184 ng/L in the water sample. This suggests that this method is appropriate for the direct analysis of ambient water samples.

references

References

1. Carmichael, W.W.; Boyer, G.L. (2016) Health impacts from cyanobacteria harmful algae blooms: Implications for the North American Great Lakes. Harmful Algae. 54, 194-212.
2. Chen, Y.; Shen, D.; Fang, D. (2013) Nodularins in Poisoning. Clin. Chim. Acta. 425, 18-29.
3. Makarewicz, J.C.; Boyer, G.L.; Lewis, T.W.; Guenther, W.; Atkindson, J.; Arnold, M. (2009) Spatial and temporal distribution of the cyanotoxin microcystin-LR in the Lake Ontario ecosystem: Coastal embayments, rivers, nearshore and offshore, and upland lakes. J. Great Lakes Res. 35, 83-89.
4. Loftin, K.A.; Graham, J.L.; Hilborn, E.D.; Lehmann, S.C.; Meyer, M.T.; Dietze, J.E.; Griffith, C.B. (2007) Cyanotoxins in inland lakes of the United States: Occurrence and potential recreational health risks in the EPA National Lakes Assessment 2007. Harmful Algae, 56, 77-90.
5. United States Environmental Protection Agency. Office of Water (4304T). Health and Ecological Criteria Division. Drinking Water Health for the Cyanobacterial Microcystin Toxins. EPA Document Number: 820R15100. (June 15, 2015).
6. Health Canada. Federal-Provincial-Territorial Committee on Drinking Water. Cyanobacterial Toxins –Microcystin-LR. (July 2002).
7. World Health Organization. Cyanobacterial toxins: Microcystin-LR in drinking-water. Background document for preparation of WHO Guidelinesfor drinking-water quality. Geneva (2003).
8. Winslow, S.D.; Pepich, B.V.; Martin, J.J.; Hallberg, G.R.; Munch, D.J.; Frebis, C.P.; Hedrick, E.J.; Krop, R.A. (2006) Statistical Procedures for Determination and Verification of Minimum Reporting Levels for DrinkingWater Methods. Environ. Sci. Technol. 40, 281-288.