Factors affecting the determination of drugs and endogenous low molecular mass compounds in human serum by micellar electrokinetic capillary chromatography with direct sample injection

Abstract

Factors influencing the establishment of an analytical window in front of the solubilized proteins in micellar electrokinetic capillary chromatography (MECC) with direct serum injection (DSI) are discussed. Both drugs and endogenous low molecular mass compounds eluting within the analytical window are identified concurrently by multi-wavelength absorption detection. Variables such as the concentration of the micelle forming substance, ionic strength, applied voltage, initial sample zone length, capillary length, selected buffer additives, insufficient renewal of the buffer in the anodic buffer vial and sample matrix are shown to impact MECC of endogenous compounds and model drugs, such as antiepileptics. For two drugs eluting within the analytical window, phenobarbital and ethosuximide, serum levels determined by DSI with external calibration are shown to compare well with levels obtained after liquid-liquid extraction and internal calibration (use of an internal standard). In addition, reproducibility of both assays is excellent. The limit of employing DSI is demonstrated with the determination of the hydrophobic drug phenytoin. Using an automated, commercial instrument and naproxen as model drug, high-speed MECC separations of high reproducibility and with a throughput of 12-15 samples per h are presented.

Micellar electrokinetic capillary chromatography as a powerful tool for pharmacological investigations without sample pretreatment: A precise technique providing cost advantages and limits of detection to the low nanomolar range

“…Due to the denaturing properties of SDS, the plasma-protein binding (PPB) is solved. Hence, bound and free drug are determined at the same time [11,33,34]. In the following years, there was an increase in the number of publications dealing with direct detection of various drugs in body fluids, including antiepileptics, nonsteroidal anti-inflammatory drugs (NSAID), antibiotics, barbiturates, etc.…”

“…In the above-mentioned earlier works, high mmolar levels have been determined with reproducibilities in the range of about 2±5% for repeatability (n & 10) and of about 4± 12% for interday precision, respectively ( [2,26], reviews in [11,30,31]; compare Table 13). Generally, CE suffers from a relatively low concentration sensitivity.…”

“…The amount of firmly adsorbed proteins was estimated to be at least 1% [42]. Second, although matrix interferences within the described ªanalyt-ical windowº are clearly reduced, there are still baseline interferences due to the presence of small endogeneous plasma components [11]. This has a strong impact on the reachable limits of detection and quantification.…”

“…Drop-outs are characterized by fluctuations of the current by more than 20%. These fluctuations lead to strong baseline irregularities which make peak integration and thus quantitative information impossible ( [9]; compare [10,11]). A systematic investigation of sodium hydroxide washings for plasma samples revealed a ratio of ³ 12% drop-outs (n = 457) [36].…”

“…Membranes with cut-offs for various molecular masses are commercially available. Problems with analyte loss due to drug binding to the serum proteins can be overcome by incubating the sample with an aqueous SDS solution prior to ultrafiltration [11,12].…”