In this study, we investigated the performance of several commercial sorbents (Sep-pack) C18, (t)C18, C8 and (t)C2, Oasis HLB, Isolute ENV+, Strata-X and Oasis MCX) for the determination of opioid peptides by solid-phase extraction coupled on-line to capillary electrophoresis (SPE-CE). First, standard solutions were analyzed in order to achieve the lowest LOD and the best electrophoretic separations using UV detection. The best results were obtained using C18, C8 and (t)C2 sorbents, which were examined for the analysis of spiked human plasma samples. A double-step sample clean-up pretreatment, which consisted of precipitation with acetonitrile and filtration, was needed to prevent saturation of the on-line SPE microcartridge. The filtration step was critical to obtain optimum analyte recovery and to clean up the sample matrix. A range of centrifugal filters and filtration conditions were tested and the recoveries of the sample pretreatment were evaluated by CE-ESI-MS. The LODs attained through SPE-CE-UV were approximately ten-fold better with C18 than with C8 and (t)C2. The 0.1 microg/mL LODs achieved by C18-SPE-CE-UV were further improved until we could detect 1 ng/mL concentrations of opioid peptides in plasma samples by C18-SPE-CE-ESI-MS, due to the outstanding selectivity of the MS detection.

One of the major drawbacks of capillary electrophoresis (CE) and other microscale separation techniques, for the analysis of low abundant peptides and proteins in complex samples, are the poor concentration limits of detection. Several strategies have been developed to improve CE sensitivity. Here, we describe an on-line solid-phase extraction capillary electrophoresis mass spectrometry method with a commercial C18 sorbent for clean-up and preconcentration of neuropeptides from highly diluted biological samples.

Fritless SPE on-line coupled to CE with UV and MS detection (SPE-CE-UV and SPE-CE-MS) was evaluated for the analysis of opioid peptides. A microcartridge of 150 μm id was packed with a C18 sorbent (particle size > 50 μm), which was retained between a short inlet capillary and a separation capillary (50 μm id). Several experimental parameters were optimized by SPE-CE-UV using solutions of dynorphin A (DynA), endomorphin 1 (End1), and methionine-enkephaline (Met). A microcartridge length of 4 mm was selected, sample was loaded for 10 min at 930 mbar and the retained peptides were eluted with 67 nL of an acidic hydro-organic solution. Using SPE-CE-MS, peak area and migration time repeatabilities for the three opioid peptides were 12-27% and 4-5%, respectively. SPE recovery was lower for the less hydrophobic DynA (22%) than for End1 (66%) and Met (78%) and linearity was satisfactory in all cases between 5 and 60 ng/mL. The LODs varied between 0.5 and 1.0 ng/mL which represent an enhancement of two orders of magnitude when compared with CE-MS. Cerebrospinal fluid (CSF) samples spiked with the opioid peptides were analyzed to demonstrate the applicability to biological samples. Peak area and migration time repeatabilities were similar to the standard solutions and the opioid peptides could be detected down to 1.0 ng/mL.

In this study, we evaluated the combination of transient isotachophoresis with on-line solid-phase extraction capillary electrophoresis time-of-flight mass spectrometry (SPE-tITP-CE-TOF-MS) to improve sensitivity of peptide analysis, using several opioid peptides as model compounds. First, standard solutions were analyzed in order to establish the tITP-CE methodology using UV and TOF-MS detection. The volume and composition of the leading and terminating electrolytes (i.e. LE and TE) for an efficient tITP were investigated to obtain optimum detection sensitivity and electrophoretic separation. In the best cases, LODs in tITP-CE-TOF-MS were tenfold better than those obtained in CE-TOF-MS (i.e. 5 versus 50 ng/mL). Afterwards, the tITP-CE-TOF-MS methodology was adapted to perform SPE-tITP-CE-TOF-MS. Repeatability, linearity and LODs were investigated and compared to the values obtained by SPE-CE-TOF-MS. Furthermore, human plasma samples fortified with the opioid peptides were analyzed in order to show the potential of SPE-tITP-CE-TOF-MS for peptide analysis in biological fluids. The LODs attained in standard solutions and plasma samples for some of the studied peptides (i.e. 0.01 and 0.1 ng/mL, respectively) were tenfold better than those obtained in SPE-CE-TOF-MS, proving the enhanced sensitivity that could be achieved when both on-line preconcentration approaches were combined together.