Advances in capillary electrophoresis: In-line preconcentration for biomedical analysis. Impurity profiling of heparin

Abstract

Capillary electrophoresis (CE) has shown to be highly suitable for the analysis of polar and ionogenic compounds in biomedical and pharmaceutical samples. Separation with CE is based on the charge-to-size ratio of analytes. The application of CE for bioanalysis may be hindered by its relatively low sensitivity. Two sample preconcentration techniques have been applied in this thesis, a dynamic pH junction and an in-capillary solid phase extraction column. In-capillary analyte stacking via dynamic pH junction was investigated with underivatised phenylalanine (Phe) and tyrosine (Tyr) as model amino acids. Samples were mixed with acidic background electrolyte prior to injection. A plug of ammonium hydroxide solution was injected before the sample to produce a pH junction. Zwitterionic amino acids have opposing charges in the respecting solvents and will thus get opposing mobilities. The amino acids stack at the interface between the solvents. LODs for Phe and Tyr in urine were 0.054 and 0.019 μM for a sample injection volume of 10% of the capillary. Another technique for preconcentration of analytes employs an in-line frit-free solid-phase extraction (SPE) column. A 2 mm * 150 µm column was packed with mixed-mode sorbent for the preconcentration of the test compounds 2-ethylidene-1,5-dimethyl-3,3-diphenylpirrolidine (EDDP), dihydrocodeine and codeine. The sorbent had a particle diameter of more than 50 µm and was retained between a short inlet capillary and a separation capillary (50 µm id). The injected sample volume was 60 µL (i.e., 30 capillary volumes). Using MS-detection, LODs were 0.22 pg/mL for EDDP, 2.1 pg/mL for dihydrocodeine and 24 pg/mL for codeine. For the analysis of opioid peptides, a similair SPE-column was packed with a C18 sorbent. The column size was 4 mm * 150 µm, and the injected sample volume was 12 µL. Using SPE-CE-MS, 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, and the opioid peptides could be detected down to 1.0 ng/mL. In 2008, oversulfated chondroitin sulfate (OSCS) present in certain lots of heparin was identified as the toxic contaminant responsible for severe side effects following intravenous heparin administration. A preliminary CE method published by the US Food and Drugs Administration showed only partial separation of the OSCS contaminant from heparin. In this thesis an improved CE method is described. Enhancement of the OSCS–heparin separation was achieved by using high concentrations of Tris phosphate (pH 3.0) as background electrolyte. Good separations of OSCS, heparin and DS are obtained within 17 min. The method permits OSCS and DS determination in heparin down to the 0.05% and 0.5% (w/w) level, respectively. The CE method was further optimized to achieve shorter analysis times. Using a much shorter capillary, separation of OSCS, heparin and DS was obtained within 7 min. Detection limits for OSCS and DS were well below 0.5 mg/mL. Further reduction of analysis time could be achieved by employing an effective capillary length of 10 cm, providing full separation within 4 min.