Page 397 - Tandem Techniques

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noted, however, that very small samples exhibited a disproportional loss of minor components, which
might make quantitative assays uncertain when operating at maximum sensitivity.

The electrospray interface has rendered the LC/MS tandem instrument available to a wide range of
applications in biology and biochemistry, which hitherto, were precluded from the technique due to its
inability to function efficiently with samples in aqueous solutions. Hua et al. [34] employed
electrospray ionization in their examination of brevetoxins. Brevetoxins are produced by the
dinoflagellate, Gymnodinium breve, and are responsible for killing fish and also pose certain health
risks to humans.

The crude brevetoxins were isolated from an extract of cultured material using a reversed phase solid-
state extraction procedure. A microbore column was employed for the separation, 10 cm long and 1 mm
I.D., packed with a C18 reversed phase, having a particle of 3 µm. The column eluent was split at a T
junction, part passing to a UV detector operating at 215 nm, and the remainder passing to the mass
spectrometer. The mass spectrometer was the Vestec Model 201 fitted with an electrospray inlet
system. However, the electrospray source was modified by replacing the nozzle used for the counter
electrode by a flat stainless steel plate with a hole in it, 0.44 mm in diameter. In addition, the 200 1 /min
pump, normally used to evacuate the first stage of the ion source, was replaced with a 500 1 /min.
pump. These modifications helped reduce the lower limit of detection by a factor of four.

The separation was developed isocratically with a mobile phase containing 15% of water in methanol,
at a flow rate of 8 µl/min. The sample consisted of 1 µl of a solution, containing 20 ng/µl (i.e. a mass of
20 ng) and the column eluent was split in the ratio 3:1, to the UV detector and the spectrometer
respectively. Good spectra were obtained and single ion monitoring produced peaks for each
component with a signal-to-noise ratio of about ten, indicating a lower sensitivity limit of less than 3
ng/ml. The significant peaks in the spectra, in fact, were sodium adduct ions of the respective
brevetoxins.