Francisco J. Lara Ana M. García-Campaña Laura Gámiz-Gracia Juan M. Bosque-Sendra Fermín Alés-Barrero Department of Analytical Chemistry, Faculty of Sciences, University of Granada, Granada, Spain Received November 23, 2005 Revised February 8, 2006 Accepted February 10, 2006 Research Article Determination of phenothiazines in pharmaceutical formulations and human urine using capillary electrophoresis with chemiluminescence detection A CE instrument coupled with chemiluminescence (CL) detection was designed for the determination of promethazine hydrochloride (PTH) and promazine hydro- chloride (PMH) in real samples. An important enhancement of the CL emission of luminol with potassium ferricyanide was observed in the presence of these phe- nothiazines; so this system was selected for their detection after CE separation. Pa- rameters affecting the electrophoretic separation were optimized in a univariate way, while those affecting CL detection were optimized by means of a multivariate approach based on the use of experimental designs. Chemometrics was also employed for the study of the robustness of the factors influencing the postcolumn CL detection. The method allows the separation of the phenothiazines in less than 4 min, achieving LODs of 80 ng/mL for PMH and 334 ng/mL for PTH, using sample injection by gravity. Elec- trokinetic injection was used to obtain lower LODs for the determination of the com- pounds in biological samples. The applicability of the CE-CL method was illustrated in the determination of PTH in pharmaceutical formulations and in the analysis of PMH in human urine, using a previous SPE procedure, achieving an LOD of 1 ng/mL and recoveries higher than 85%. Keywords: Capillary electrophoresis / Chemiluminescence / Pharmaceutical analysis / Phenothiazines / Urine analysis DOI 10.1002/elps.200500863 1 Introduction CE is a useful analytical technique in different fields such as proteomics [1], pharmaceutical [2], pesticides analysis [3], etc. Its main advantages are high efficiency and reso- lution, the low reagent and sample consumption, and short analysis times. However, its main drawback is the lack of sensitivity because only a few nanoliters of sample are introduced in the capillary. There are some strategies to obtain lower detection limits such as the application of on-line preconcentration techniques [4, 5] or the use of more sensitive detection systems, such as luminescence methods. Among them, LIF [6–8] has been widely used but chemiluminescence (CL) remains still unfamiliar al- though it has proved to be, in general, very sensitive in both flow-injection analysis (FIA) and HPLC because of its low background nature [9]. When combined with a ver- satile and efficient separation method such as CE, CL can offer excellent analytical sensitivity [10, 11] and selectivity [12], allowing the resolution and quantification of various analytes in complex mixtures. In this sense, the coupling CE-CL could be a powerful strategy to resolve problems where extrasensitivity and also high efficiencies are needed or when reduced quantities of sample are avail- able [13, 14]. Various CL reagents have been used in CE- CL, mainly peroxyoxalates, ruthenium complexes in the case of electrogenerated CL (ECL), and luminol and some derivatives as isoluminol isothiocyanate or N-(4-amino- butyl)-N-ethylisoluminol (ABEI). To a less extent, direct Correspondence: Dr. Ana M. García-Campaña, Department of Ana- lytical Chemistry, Faculty of Sciences, University of Granada, Avd. Fuente nueva s/n, E-18071 Granada, Spain E-mail: amgarcia@ugr.es Fax: 134-958-249510 Abbreviations: CL, chemiluminescence; IS, internal standard; PMH, N,N-dimethyl-3-(10H-phenothiazin-10-yl)propan-1-amine; PTH, N,N-dimethyl-1-(10H-phenothiazin-10-yl)propan-2-amine; TMS, N,N,N,a-tretramethyl-10H-phenothiazine-10-ethanaminium methyl sulfate 2348 Electrophoresis 2006, 27, 2348–2359  2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com