Ion trap tandem mass spectrometry of biological molecules using wide range excitation CID Product ion spectra obtained by ion trap tandem mass spectrometry (MS n ) are often very simple owing to the low-energy characteristic of the resonant excitation used for collision-induced dissociation (CID). 1 This is sometimes disadvantageous for the analysis of biological molecules, many of which exhibit multiple low-energy fragmentation pathways such as neutral water losses. Consequently, these product ion spectra then give only limited structural information such as molecular masses or the presence of OH and carboxylic groups. Sequential MS n can often be performed to obtain more structure information from the molecules of interest. However, there is a limitation: isolation and CID efficiencies usually range from 50 to 90%, 2 limiting the use of higher order multiple-stage fragmentations for low-concentration environmental and biological samples. To overcome this limitation, the latest generation of commercial ion-trap mass spectrometers allow the excitation of single or multiple mass range(s) 3,4 in order to fragment simultaneously both the precursor and fragments formed by neutral losses such as water. For example, the recent version of the ThermoFinnigan LCQ is able to bring ions in a mass range of 20 u below the selected precursor ion into resonance excitation by defining an excitation frequency and ramping the r.f. voltage applied to the ring electrode. 3 This 'wide-band' excitation provides 'richer' fragment ion spectra (so-called pseudo-MS 3 ) of appropriate analytes. 5 However, it only allows simultaneous fragmentation of one water loss product ion.
The aim of this contribution is to address the optimization steps necessary to obtain reliable wide-range excitation CID spectra on an unmodified commercial ion trap. The biotoxin class of azaspiracid and its analogs was chosen to demonstrate the usefulness of the advanced wide-range excitation because ion-trap CID of these compounds results in consecutive losses of up to six water molecules without showing significant fragments in the low-mass range. These polyether compounds are found in shellfish and contain unique spiro-ring assemblies, a cyclic amine and a carboxylic acid (see Fig. 1). Detailed discussions of their origin, toxicity and mass spectral dissociation behavior are given elsewhere. 6 -10 In contrast to ion-trap MS 2 , triple-quadrupole (TQ) MS 2 gives significantly more structural information (see Fig. 1 and Brombacher et al. 10 ). The described procedure overcomes the need for sequential MS n when several water molecules are primarily cleaved from the precursor ions and allows one to obtain TQ-like CID product ion spectra. The authors hope that this contribution will help other mass spectrometrists to use this method in their applications.
The experimental setup included an Agilent (Palo Alto, CA, USA) 1100 high-pressure binary gradient HPLC system and a PurospherStar column (C 18 , 2.0 รฐ 50 mm, 3 ยตm particles; Merck, Darmstadt, Germany). The gradient (flow-rate 350 ยตl min 1 ) used was acetonitrile-water (50 mM formic acid, 2 mM ammonium formate) (50 : 50), isocratic for 1 min, then in 7 min to 100% acetonitrile, with an isocratic hold for 3 min. The crude mussel tissue extract used in this study (provided by the FRS-Marine Laboratory, Aberdeen, UK) contained five azaspiracids (AZA 1-5; AZA 1 at ยพ0.2 ยตg ml 1 ). Electrospray data were acquired with an Agilent LC/MSD Trap SL ion-trap mass spectrometer in the positive ion mode: spray voltage, 3.5 kV; nebulizer gas, 3.1 bar; dry gas (325 ยฐC) flow, 7 l min 1 . TQ product ion spectra were acquired on an API 4000 instrument (MDS-Sciex, Concord, ON, Canada) with a collision-offset voltage of 60 V and nitrogen as collision gas (pressure setting: 6).
The ion trap used in this study allowed the simultaneous resonance excitation of the z-axial secular frequencies of an m/z range of up to 300 u (ลก150) centered on the precursor ion without