Victoria Sanz-Nebot Fernando Benavente Elvira Balaguer José Barbosa Departament de Química Analitica, Universitat de Barcelona, Barcelona, Spain Capillary electrophoresis coupled to time of flight-mass spectrometry of therapeutic peptide hormones We have established a method for separation and characterization of a series of peptide hormones of pharmaceutical interest and wide therapeutical use by capillary electrophoresis-electrospray-mass spectrometry (CE-ES-MS) using a sheath flow interface. Several parameters were systematically investigated, such as concentration of the electrolyte, organic solvent and sheath liquid composition, gas flow rates and capillary position. Moreover, limits of detection, linearity, repeatability and day-to-day reproducibility of the proposed method were studied in order to obtain the main quality parameters. Keywords: Capillary electrophoresis / Hyphenated techniques / Peptide hormones / Time of flight-mass spectrometry EL 5337 1 Introduction Capillary electrophoresis (CE) has become a powerful analytical technique with high analysis speed, high separation efficiency and low sample amounts needed [1]. This technique is widely used for analysis of a high number of biomolecules as peptides and proteins [2–5]. The coupling of CE with a mass spectrometer (CE-MS) provides a powerful system for the analysis of complex biological mixtures, replacing or complementing other conventional detection methods such as absorbance, electrochemical detection or laser-induced fluorescence, which prove to be less informative and universal [1, 6]. The use of a mass spectrometer as a detector enhances the utility of the CE and allows an efficient separation and identification of components in complex mixtures, obtaining structure and/or molecular mass information [1]. Among all kinds of mass spectrometers that can be used in combination with CE, a time-of-flight (TOF) ana- lyzer is an ideal detector [1, 7, 9]. One advantage of this kind of analyzer is the fast acquisition rates (0.1 s) that provides an extremely short time to produce a full mass spectrum [7], allowing a high number of spectra for time unit and the complete characterization of the peak profile. Other advantages of a TOF analyzer are good mass resolution and sensitivity because most of the ions introduced into the vacuum system reach the de- tector [8]. The most used ionization technique for coupling CE and MS is electrospray (ES). Advantages of this technique are its high ionization efficiency, the soft nature of the ioniza- tion process, which avoids undesired fragmentations, the multiple charged ions obtained, which provides an m/z range compatible with most of the analyzers used, and the direct transference of molecules from the liquid phase to the gas phase via a CE-ES-MS interface, which pro- vides a stable spray and current [6, 9–10]. Although three kinds of interfaces, sheathless, liquid-junction, and sheath-flow interface, have been used for CE-ES-MS coupling, sheath-flow interface has predominately been applied because it has an easy and reproducible con- struction, and allows a great versatility for buffer choice [6, 8, 10]. In this design the CE capillary is introduced in a steel tube, where the sheath liquid, usually a hydroorganic mixture, is also delivered. The tip of the tube is where the mixing of the sheath liquid and the separation buffer takes place. Thus, the CE flow rate is increased in several microliters per minute, improving spray stability but dilut- ing the capillary eluent [8]. The sheath liquid must contain an electrolyte because it also acts as a CE terminal buffer allowing the electrical continuity and an efficient electro- phoretic separation [6, 10]. In the last years, CE-ES-MS has been principally used for the analysis of peptides, peptide hormones and proteins, drugs and drug metabolites, biological extracts and for environmental analysis [1, 6]. Among these wide appli- cation fields, peptides represent a numerous class of important and widely spread biomolecules. Acting as hor- Correspondence: Dr. Victoria Sanz-Nebot, Departament de Quí- mica Analitica, Universitat de Barcelona, Diagonal, 647, E-08028 Barcelona, Spain E-mail: vsanz@apolo.qui.ub.es Fax: + 34-93-4021233 Abbreviations: MeCN, acetonitrile; MeOH, methanol; TIE, total ion electropherogram; XIE, extracted ion electropherogram Electrophoresis 2003, 24, 883–891 883  2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 0173-0835/03/0503–883 $17.501.50/0 CE and CEC