An interview with Rohan Thakur, discussing the prevalence of microcystins in drinking water, and the use of LC-MS to identify cyanotoxins within water samples.
What are microcystins and where are they found?
Microcystins have become the generic name for the toxins produced from blue-green algae which can bloom in surface water impoundments, slow-moving streams, and rivers. Microcystins are the most common of the cyanobacterial toxins found in water, as well as being the ones most often responsible for poisoning animals and humans who come into contact with toxic blooms.
The name microcystin originated from the discovery of a specific toxin isolated from cyanobacterium called Microcystis aeruginosa. Chemically they are cyclic heptapeptides.
Are there different types of microcystin and are they all harmful to humans?
Microcystins and other related cyanobacterial toxins can be generally grouped into three categories, hepatotoxins (attacking the liver), neurotoxins (attacking the nervous system), and others that tend to be skin and membrane irritants.
Of these, the hepatotoxins are of most concern due to liver problems caused by acute exposure and potential carcinogenic issues related to chronic exposure.
Of less concern today are the potential neurotoxic and irritant effects. However, less than 100 toxins have been identified, so there is limited data on the types and categories of all toxins related to cyanobacteria.
By what mechanism do microcystins cause harm to humans?
The blue-green algae can form in waters that have high nutrient levels, e.g. nitrogen and phosphorus. Agricultural run-off carrying phosphorus fertilizer or manure into the surface water impoundment, creek or river is a primary cause of the excess nutrient conditions. Excess nutrients, cyanobacteria and appropriately warm temperature will cause an algal bloom to form.
As the blue-green algae die their cell walls decay and release the stored toxins. Many of these toxins are quite stable in water and can remain intact for extended periods of time.
Human exposure results from drinking untreated or poorly treated water (boiling the water will not eliminate the toxins), consuming fish that have bio-accumulated the toxins, or livestock that drink from ponds.
Illness and death occur depending on the type and quantity of the toxin ingested. Microcystins target the liver reducing function to a level causing illness and potentially death.
Proper water treatment will eliminate the threat of microcystins exposure. Unfortunately for many developing countries and rural areas that rely on shallow wells or surface water, the threat is present for both humans and livestock.
What levels of microcystin are toxic to humans?
Currently there is no study which has documented toxicity in humans. Animal studies for mice have reported LD 50 for oral administration at 5 mg/kg. Different countries have set regulatory levels in drinking water at 5 µg/l or 5 ppb. The World Health Organization (WHO) has set the limit at 1 ppb (1 µg/L).
How are microcystins analyzed?
The method of choice is LC-MS/MS on triple quadrupole mass spectrometers run in MRM (multiple reaction monitoring mode). Typically one will monitor the presence and levels of specific target microcystins as indicators for the presence of toxins.
Please can you tell us about Bruker’s EVOQ EliteTM liquid chromatography mass spectrometer triple quadrupole (LC-MS/MS)?
The EVOQ Elite is targeted at LC-TQ performance leadership and major advances in robustness, ease of methods development and productivity. It was purpose-built for sustained high-sensitivity quantitative analysis applications.
It also has several innovations such an industry first, orifice-linear dual ion funnel interface that delivers the high-sensitivity and robustness; and the VIP-HESI ion source, which effortlessly ionizes thermally labile molecules.
This was particularly critical for the high-sensitivity analysis of microcystins, which are cyclic peptides and thermally labile. Using the VIP-HESI interface, sub-ppb levels of microcystins could be easily quantified using minimum sample preparation.
Other high-performance TQ features include Bruker’s unique Compound Based Scanning (CBS) technology, APCI probes standard, fast 14,000 amu/sec scan speed, and 25 msec positive/negative ion switching, all for leading-edge TQ performance and analytical power.
How sensitive is the EVOQ Elite LC-MS?
The EVOQ can easily achieve detection limits well below the 1 ppb action level. We have an application note that shows detection limits of 50 ppt or 0.05 ppb.
While sensitivity is the key for this application - if you cannot detect at 1.0 ppb you are out of the game - laboratories are just as keen on sustained sensitivity. These labs may run hundreds to thousands of samples, per day per week etc. If the instrument has great sensitivity but cannot sustain that performance for more than between 20 and 30 samples before required maintenance, then the lab will suffer from poor productivity. This costs time and time is money.
How does the EVOQ Elite LC-MS differ from other liquid chromatography mass spectrometers that are currently available?
The EVOQ system was developed to provide excellent sensitivity and industry leading sustained sensitivity. Key design elements in the system provide incredible robustness that allows users to run literally thousands of samples before required cleaning or maintenance.
A couple of examples, typical source contamination is caused by inefficient exhaust of the LC effluent from the API source housing. This allows build-up of residual material in the source housing that will increase noise in the system and reduce ion transmission into the mass spectrometer. By focusing our attention to this area, our scientists were able to provide an innovative solution that produces tremendous increase in system uptime.
The EVOQ system allows the user to focus on solving the analytical problem, and not be distracted by the instrument parameter operation.
The EVOQ greatly simplifies MRM-based quantitation with Bruker’s unique Compound Based Scanning (CBS), which makes MRM methods development trivial, and MS/MS as easy to use.
A key advantage is the orifice interface, which lends itself to be highly cost-effective, while providing the requisite sustained ultra-high sensitivity advantage when compared to capillary-based LC-TQ’s.
Replacing these ion transfer capillaries gets expensive over the life of the instrument, and they are prone to frequent clogging. The orifice ion source coupled to the Active Exhaust system provides exceptional robustness, while allowing for sustained ultra-sensitivity quantitative analysis.
Can the EVOQ Elite LC-MS be used in diagnostic procedures or should it only be used for research?
The EVOQ is not a diagnostic instrument. The data that is generated would be used in research and production laboratories. A trained analyst would then use the information and make decisions, judgements etc.
Where can readers find more information?
About Rohan Thakur
Rohan Thakur is currently Vice President of the Chemical and Applied Markets division at Bruker Daltonics, based in Fremont, California, US.
Rohan has global experience in the field of life sciences mass spectrometry solutions for the pharmaceutical drug discovery and development, CRO industry, clinical chemistry, environmental, food safety, metabolism and metabolomics markets, including informatics solutions such as Watson LIMS.
Prior to his role at Bruker Daltonics, Rohan was Executive Director for Strategic Initiatives at PharmaNet Development Group, Inc. Before this Rohan was Associate Director for Specialized Mass Spectrometry at Taylor Technology.
Rohan studied at Harvard Business School, University of Medicine and Dentistry of New Jersey - Robert Wood Johnson Medical School, Kansas State University.
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