Accurate LC-MS analyses for microcystins using per-15N-labeled microcystins

Abstract

Per-(15)N-labeled microcystins were prepared for use as surrogates for accurate liquid chromatography-mass spectrometry analysis. Two strains of Microcystis aeruginosa were cultured in (15)NO(3)-containing TS-15 medium. To change from the incorporation of (14)N to (15)N into all cell components, cells of Microcystis aeruginosa were precultured in Na(15)NO(3)-containing medium for more than 6 months. After mass cultivation of the strains, cells of each strain were harvested and lyophilized. Microcystin variants were extracted from the lyophilized cells and per-(15)N-labeled microcystin variants were purified using high-performance liquid chromatography and high-performance thin-layer chromatography. The structures of per-(15)N-labeled microcystin variants were confirmed by their mass spectrometry spectra and NMR spectra. When per-(15)N-labeled microcystins were used as surrogates for quantitative analysis of these toxins in cyanobacterial cells, excellent accuracy (98-106%) was obtained, with the m/z of M(+), [M+1](+), and [M+2](+) of both microcystins and the per-(15)N-labeled microcystins as surrogates being completely separated. In conclusion, per-(15)N-labeled microcystins are excellent surrogates for microcystin analysis using liquid chromatography-mass spectrometry.

Isotope-labelling derivatisation: a broadly applicable approach to quantitation of algal toxins by isotope dilution LC-MS/MS

“…The fragmentation of the dansylated DA derivative has been described previously and is dominated by a cleavage at the sulfonate bond in the DNS moiety to form the dimethylaminonaphthalenium product ion at m/z 170. Although less expensive and more accessible, the drawback to using deuterium rather than 13 C-or 15 N-labelling for isotope dilution is that it can alter chromatographic behaviour. This was the case when pairs of d 0 -and d 6 -isotopologues of DA, ATX and hATX were analysed using standard RP-LC separation conditions ( Fig.…”

“…3B). Future work to synthesise 13 C-labelled dansyl chloride, as recently reported, 14 could be carried out to eliminate these isotope effects and allow for more selective separation to be used in the future.…”

“…Though more readily available than reagents labelled with 13 Co r 15 N, deuterium-labelled DNS does introduce isotope effects into chromatographic separations. We demonstrate how these effects can be minimised in the case of d 0 /d 6 -dansylated toxins by manipulating the selectivity or retention characteristics of the LC separation.…”

“…Future work will be directed towards expanding the utility of this approach for algal toxin analysis by LC-MS. This will include expanding the approach to other toxin classes where matrix effects have a greater potential to hinder accurate quantitation by LC-MS. Also of interest will be to move away from the use of deuterated reagents towards the synthesis of 13 C and 14 N labelled derivatisation reagents in order to minimise the impact of labelling on chromatographic separations. The approach used here of derivatising a calibration solution with the labelled reagent along with each sample set is shown to be an effective method of quantitation.…”

A High Accuracy and Simple Analytical Method for Microcystins Using ¹⁵N－labeled Microcystins

“…This fact means that the condensation procedure using solid phase extraction is not necessary. In addition to improve the accuracy, we developed a method using stable isotope ( 15 N)-labeled microcystins as sur rogates for correction of the recoveries and ionization efficiencies 7) . The accuracy of LC-MS/MS analysis for microcystins was greatly improved by using of 15 N-microcystins as surrogates.…”