Report No FR0224

H A James and C P James

Oct 1991



  1. To develop a reproducible analytical method which will enable microcystin-LR levels in reservoir and drinking waters at concentrations down to 100 ng/l to be monitored.
  2. To investigate mass spectrometric based methods for definitive confirmation of the presence of microcystin-LR in extracts of reservoir and drinking water samples at the level of interest in 1.


Blooms of blue green algae have in recent years increased in prevalence in UK waters, and under certain conditions many blooms produce toxins. Microcystis aeruginosa, which occurs widely, produced several hepatotoxins, the commonest of which is microcystin-LR. This is of high mammalian toxicity, and it is vital to be able to detect and quantify this toxin in reservoir and drinking waters.


This report has been produced in three parts. The first part relates to the development of the method, the second (which forms the Appendix to this report) gives details of the experimental work carried out, and the third part (the finally developed method) has been produced as a separate Annex to the report, so that the details necessary to allow the method to be established are readily accessible.

Much of the experimental work involved assessing the utility of various types of solid phase cartridges for the extraction of microcystin-LR from water samples and investigating different potential detection methods. Interferences present in both reservoir waters and drinking waters initially presented problems, but it eventually proved possible to reduce these to acceptable levels.

Investigation of the performance of the method by spiking microcystin-LR and the internal standard used (nodularin) into samples of Grafham Water and treated Grafham Water gave a limit of detection of less than 100 ng/l for treated Grafham Water, and a detection limit of about 200 ng/l for Grafham Water. The reproducibility of the method is good, with coefficients of variation of about 10% for both reservoir water and drinking water at microcystin-LR levels above 1 g/l. Confirmation of mirocystin-LR levels above 2 g/l can be obtained by using fluorescence detection following a post-column derivatisation which is specific for arginine-containing peptides.

Electrospray mass spectrometry can be used to confirm the presence of microcystin-LR in extracts of reservoir and drinking water provided that the level present in the water samples is >1 g/l. The level that can be detected using mass spectrometry appears to be limited by interferences present in the extracts, as much lower levels can be detected when standard solutions are examined.


A reproducible method has been developed which allows low levels of microcystin-LR to be monitored in reservoir and drinking waters. It is possible to confirm these levels (provided they are >1 g/l) using electrospray mass spectrometry.


The method finally developed, described fully in the Annex to this report, needs to be tested in other laboratories to confirm that the performance achieved with Grafham Water and treated Grafham Water is generally applicable.


The method developed involves solid-phase extraction and clean-up, using weak cation exchange cartridges, and HPLC analysis with detection/quantification using ultra-violet (UV) followed by fluorescence detection (after a post-column derivatisation reaction which is specific for arginine-containing peptides).

Investigations of the performance using samples of Grafham Water and treated Grafham Water indicate that the method with only fluorescence detection is satisfactory for levels of microcystin-LR in the range 2-50 g/l in reservoir waters, but that due to interferences (thought to be humic materials) it is difficult to detect levels <1 g/l. With UV detection, levels of less than 200 ng/l can be detected in reservoir derived drinking water, and levels less than 1 g/l can be detected in reservoir water. The limits of detection derived from calibration curves produced with UV detection are 50 ng/l and 205 ng/l respectively. In the range 2-50 g/l for Grafham Water and 1-50 g/l for treated Grafham water, the coefficients of variation were <10%. At the lowest spiking levels (1 g/l for Grafham Water samples, and 0.2 g/l for treated Grafham Water), the coefficients of variation were 23% and 26% respectively.

The method has not been validated for other reservoir or lake waters, or other treated waters, and it is suggested that prior to using the method, some validation work is carried out using appropriate water samples.

Electrospray mass spectrometry proved to be extremely sensitive for standard solutions of both microcystin-LR and nodularin, but the response obtained from direct examination of water extracts was adversely affected by interferences present, and the detection limit for confirmation by mass spectrometry is about 1 g/l.


A method based on solid phase extraction and analysis using high performance liquid chromatography (HPLC) with either ultra-violet (UV) or fluorescence detection (or a combination of both), has been developed. By using an internal standard, nodularin, the reproducibility of the method is good. The limit of detection for drinking water samples is below 100 ng/l, and is about 200 ng/l for the reservoir water samples (Grafham Water) used for method validation studies.

Electrospray mass spectrometry appears to be a suitable technique to confirm the levels found by HPLC analysis with UV or fluorescence detection, provided that the levels are >1 g/l. Nodularin is a suitable internal standard for quantification using mass spectrometry.

The method has been specifically developed for microcystin-LR. With UV detection only, the presence of a peak on a HPLC chromatogram at the correct retention time (i.e. the same retention time as obtained from a standard of microcystin-LR) strongly suggests that it is microcystin-LR that has been detected. The certainty of detection is increased if fluorescence detection is used (either alone, or following the UV detector), and provided the level detected is >1 g/l unequivocal confirmation is provided by mass spectrometry.

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