Journal of Chromatography B, 800 (2004) 315–319 Reversed-phase liquid chromatographic–mass spectrometric determination of microcystin-LR in cyanobacteria blooms under alkaline conditions Werawan Ruangyuttikarn a,∗ , Ivan Miksik b , Jeeraporn Pekkoh c , Yuwadee Peerapornpisal c , Zdenek Deyl b a Department of Forensic Medicine, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand b Institute of Physiology, Academy of Sciences of the Czech Republic, Czech Republic c Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai, Thailand Abstract Reversed-phase HPLC coupled to the atmospheric pressure ionization–electrospray ionization (API–ESI) MS was used for microcystin-LR detection and quantitation in samples of dried Microcystis aeruginosa cells. An alkaline linear gradient (20 mmol/l ammonium hydroxide– acetonitrile, pH 9.7) was used for elution of the toxic peptides. Limit of detection was 1 g/ml (20 ng per injection) in the scan mode of MS and 0.1 g/ml (2 ng per injection) in the case of selective ion monitoring. © 2003 Elsevier B.V. All rights reserved. Keywords: Cyanobacteria; Microcystin 1. Introduction Toxic cyanobacteria, or blue-green algae, represent a serious problem in freshwater reservoirs in northern Thai- land where all these reservoirs are contaminated with toxic cyanobacteria, Microcystis aeruginosa Kutz. [1]. Even when the toxins present (cyclic heptapeptides known as mi- crocystins) do not exceed the WHO water guideline value (1 g/l) for microcystin-LR [2], they still represent a serious health risk for the residents [3]. Owing to their hepatotoxic and tumor promoting activity [4–6], it was proposed that these compounds are responsible for the increased incidence of hepatocarcinoma in exposed populations, in particular in developing countries, where a high proportion of the population consumes untreated surface water [7,8]. All microcystins possess a common cyclic heptapeptide structure, contain unusual amino acids and their hepatotoxi- city is based on their capability to inhibit liver phosphatases [9]. This effect is due to the presence of a hydrophobic amino acid, 3-amino-9-methoxy-10-phenyl-2,6,8-trimethyl-deca- 4,6-dienoic acid (ADDA), in their structure, which binds to the same site on the enzymes as okadaic acid and may, ∗ Corresponding author. therefore, act as a tumor promotor [2]. Over 50 microcystin analogues have been isolated and identified so far [10,11]. A number of analytical techniques are currently avail- able for both the quantification and identification of mi- crocystins in naturally occuring samples: enzyme based assays [12], immunoassays [13,14] (inclusive immunoaffin- ity purification [15]), thin-layer chromatography [16] and high-performance liquid chromatography techniques [17–20] (also in combination with mass spectrometry [21]) can be traced in the literature. Capillary electrophoresis (occasionally coupled to MS) represents another method of choice [22–24]. In all methods reported increasing of the sensitivity and the possibility to detect (and quantitate) minute amounts of the toxins was emphasized. Of particular interest is the quantitation of microcystin-LR which is the most abundant and most toxic member of the microcystin family of peptides. In the case of analyzing Microcystis aeruginosa cells, sensitivity of the method used is not of prime importance as the microcystin content in this material exceeds considerably the concentrations found in polluted water. As far as the liquid chromatography methods for mi- crocystin analysis is concerned, all of them used an acidic (acetic or formic acid) based mobile phase. The peptide or protein analysis is routinely made in mobile phases with 1570-0232/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/S1570-0232(03)00595-6