Influence of mobile phase conditions on the clean-up effect of restricted-access media precolumns for plasma samples injected in a column-switching system

Abstract

SummaryVarious mobile phases including phosphate buffer, pure water and five kinds of biological buffers (pH around 7) were systematically studied in terms of their ability to clean-up plasma matrix on precolumns of restrictedaccess media in a column-switching system. The necessary washing time, buffer pH, type and content of organic modifier were evaluated with respect to plasma elution profiles on restricted-access media precolumns. The influence of different mobile phases on the recovery of plasma matrix from alkyl-diol silica precolumns was studied by means of a scanning spectrophotometer. Our results show that phosphate buffer near physiological pH with small amounts of 2-propanol or acetonitrile was preferable for direct injection of large plasma volumes (500 gL). More than 93 % of the proteins in a plasma matrix can be recovered within 3 min from the alkyl-diol silica C18 column (25 mx 4 mm I.D.) as measured at 280 nm for all selected mobile phases except for tris (hydroxymethyl) aminomethane buffer, from which only about 88 % was obtained.

Desenvolvimento de colunas cromatográficas de meios de acesso restrito proteína-imobilizada e suas avaliações para análise de fármacos com injeção direta de plasma humano

“…O uso de fases móveis em pH pró-ximo ao ponto isoelétrico (pI) da albumina leva a uma perda de performance de exclusão e à precipitação das proteínas do plasma. Portanto, não é recomendável o uso de eluentes com pH entre 3 e 5 em métodos analíticos por injeção direta de amostra que utilizam colunas RAM 31 . A Figura 6 mostra as variações do fator de retenção dos fármacos avaliados em função do pH da fase móvel.…”

High-performance liquid chromatographic determination of cocaine and benzoylecgonine by direct injection of human blood plasma sample into an alkyl-diol-silica (ADS) precolumn

“…This means that the macromolecules such as the protein of the human blood plasma sample can be excluded in the void volume to waste. Additionally, the inner surface of the porous particles is covered with hydrophobic C-18 phase and these adsorption centers are freely accessible to the analytes of low molecular weight [17,18,19,20]. In order to determine the elution profile of the sample matrix and analytes the ADS extraction precolumn was first directly coupled to the UV diode-array detector set at 235 nm and the composition and flow-rate of the extraction mobile phase were studied.…”

“…However, direct injection technique is generally preferable, since problems involved in off-line sample pretreatments, including time-consuming procedures, errors and the risk of low recoveries, can be readily avoided. In this way, alkyl-diol-silica (ADS) precolumns, which were previously introduced by Boos et al [17] and belong to a family of restricted-access materials (RAM), have been successfully used for the determination of low-molecular-weight compounds in body fluids by direct injection [18,19,20].…”

Characterization of the precolumn bio trap 500 C18 for direct injection of plasma samples in a column-switching system

“…However, the presence of 2 mM tetrabutylammonium seems to increase the solvation of the proteins in phosphate buffer. Acetonitrile and 2-propanol have earlier been shown to play a similar role on protein recovery [23]. It is further seen that the plasma proteins tend to retain on the BioTrap surface when using TRIS buffer, pH 7.4.…”