Short Communications z On-Line Preconcentration-Capillary Electrophoresis-Mass Spectrometry (PC-CE-MS) Andy J. Tomlinson,* Linda M. Benson, W. David Braddock, Robert zyxwv P. Oda Biomedical Mass Spectrometry Facility and Department zyxwvutsrq of Biochemistry and Molecular Biology Stephen Naylor* Biomedical Mass Spectrometry Facility and Department of Biochemistry and Molecular Biology and Department of Pharmacology, Guggenheim COO9B, Mayo Clinic, Rochester, MN 55905, USA Key Words: Capillary electrophoresis Electrospray mass spectrometry Sample concentration Peptides Drug metabolites 1 Introduction A perceived limitation of capillary electrophoresis (CE) is the ability to load large volumes of dilute solutions contaming biologcally active compounds onto the capillary while maintaining resolution of components.This has been overcome,to some extent, by the use of transient isotachophoresis (tITP)[1,2], field amplification 131,and analyte staclung [4,5]. More recently, we have been investigating the application of preconcentration-CE (PC-CE) using a cartridge containing a small quantity of solid phase packing material zyxwvuts (e.g., C-18 reversed phase) on-line with the CE capillary zyxwvu [6,7]. These PC-CE capillaries are similar to those described by Guzman [8], and Swam and Merion [9] We have also been exploring the use of PC-CE in conlunction with on-hne CE-mass spectrometry (PC-CE-MS). In the past, the prob- lems of sample loading have been reported to be circumvented by tITP-CE-MS [lO,ll]. In this work we report, to our knowledge, the first use of on-linePC-CE-MSfor the analysis of mixtures containing structurally similar compounds for both putative drug metabolites and peptides. 2 Experimental 2.1 Chemicals Acetic acid (99 9% + grade) was obtained from Aldnch Chermcal Company (Milwaukee, WI, USA) and HPLC grade methanol and water were obtamed from Baxter (Minneapohs, MN, USA) and shca-based C-18 (irregular particles) from Waters (Milford, MA, USA) Halopendol and the synthetic analogs were a gift from Pro- fessor J W Gorrod (Universityof London, UK) The peptides SGIN- FEKL, SIINFEKLT,and LSPFPFDL were synthesized in the Protein Core Fachty at Mayo Chnic 2.2 PC-CE-MS The preconcentration capillary used in these experiments was prepared from uncoated fused sihca tubing (50 pm i d ) pretreated with sodium ethoxlde and methanol A 2 mm packed bed of dry end-capped sihca C-18 matenal was packed in Teflon tubing zyxwvu (ca 400 pm i d ) and connected to the treated shca capillary 1 5 cm from the end of the inlet The inlet of the CE capillarywas extended to full length by the addition of 1 5 cm of treated fused sihca tubing The final dimensions of the capillary were 50 pm i d x 70 cm in length The entire PC capillary was then conditioned under high pressure (20 psi) for ten minutes each with methanol, water and separation buffer respectively zyx All subsequent capillary treatments and sample loading washing, and elution were also carried out under h g h pressure The capillarywas conditioned with separation buffer (see individual figure legends) containing 3 pM halopendol or 10 pM peptide LSPFPFDL for 30 min Pnor to analysis the capillary was nnsed with separation buffer (5 min) and methanol (0 5 mm) to remove excess conditioning compound The system was equhbrated in separation buffer (5min) Methods of analysis included a cleaning regime of methanol (0 2 rmn) and separation buffer (5 min), followed by a high pressure inlection of the respective mixture (30 s inlections) The capillary was then washed with separation buffer for 5 min and the analytes were eluted off the paclong matenal with methanol (halopendol mixture) or 90% methanol 10%separation buffer (peptide mixture) followed by a plug of separation buffer (0 3 min) CE separations were performed using a modified Beckman PIACE 2100 instrument (Fullerton, CA,USA) coupled to a Reason Technol- ogy 486 PC (Rochester, MN, USA) with system control and data capture by System Gold software (Beckman,Fullerton, USA) The CE separation medium used to afford optimum separation of the mixtures is described in the Figure legends. All analyses were carried out on a Finnigan MAT 900 mass spectrometer (Bremen, Germany) of EB configuration (where E is an electric sector and B is a magnet) with a PATRIC (positionand time resolved ion counter) focal plane detector. Either a modified Analytica (Banford, CT, USA) or a Finnigan MAT (Bremen, Germany)electrosprayion source was used in positive ion mode. In the former case, the needle assembly is at ground and the Nz drylng gas was set at 140°Cwith a flow rate of 3.6 literstmin and an ESI voltage of 3200V. The Finnigan MAT source employs a spray needle that is floated to voltage (typically 6-8 kv) and a heated metal capillary (200°C) as the first stage of separation of the atmospheric pressure spray regon from the vac- uum of the mass spectrometer. Neither auxillary nor sheath gas were used with the Finmgan MAT source. All experiments were conducted at a resolution of ca.1000using an 8% PARTIC window, scanning at 2 stdecade.The mass range scanned for the haloperidol analysis was 125-450 da and for the peptide analysis 300-1300 da. Journal of High Resolution Chromatography VOL. 17, OCTOBER 1994 729