A study of column switching liquid chromatography in drug analysis.

Alexander, Christine

Abstract

Drug analysis methods and the sample pretreatment of biological fluids are reviewed. A survey of column switching liquid chromatography techniques revealed the range of column dimensions, the frequency of prior sample pretreatment steps and small injection volumes used and the varied longevity of the columns. The advantages and disadvantages of microbore columns and their use in column switching systems are discussed. A systematic study was carried out with 4.6, 2 and 1 mm analytical columns and varied precolumn dimensions. The mass sensitivity of six steroids, differing widely in hydrophobicity, increased with decreasing analytical column diameter. Efficiency, sensitivity and resolution decreased on addition of the precolumn into the system with the 1 mm analytical column only, the variation between precolumns being slight in comparison. To investigate sample clean-up, the study was extended to analyse steroids in biological fluids. Saliva and urine analyses were successful, provided adequate separation from the large urine front was achieved. Linear calibrations were obtained with plasma solutions, although efficiency, sensitivity, recovery and longevity of the precolumn were reduced in comparison to aqueous solutions. Recovery was dependent on the concentration of plasma in the sample and was lower with the smaller 1x10 mm precolumn. Irreversible adsorption of matrix components in the column adversely affected the performance of the system. A column switching assay for biguanides involving an analytical mobile phase containing a high concentration of hydrophobic pairing ion was developed and validated. The steady state concentrations of proguanil and metabolites were determined in plasma and saliva, obtained from a study involving ten volunteers. Due to the low concentration of biguanides detected in saliva and lack of correlation between saliva and plasma samples, saliva monitoring can not be recommended for determining the potential efficacy of the antimalarial. An assay method based on on-line sample pretreatment was developed to analyse trioxsalen, which is strongly protein bound. A detection limit of 5 pg ml-1 was achieved with aqueous solutions but was severely restricted to 10 ng ml-1 in plasma solutions. After trioxsalen bath therapy plasma contains very low concentrations of trioxsalen, and lack of sensitivity due to sample clean-up inhibited the detection of trioxsalen in patient samples. General conclusions are drawn as to the overall utility of column switching liquid chromatography in drug analysis. Column switching with microbore columns was successful, although extra column band broadening limits the increase in mass sensitivity. Column switching may be used when a high concentration of ion pairs is necessary for separation. On-line sample pretreatment is matrix dependent and lack of effective sample clean-up may prevent the preconcentration and sensitivity advantages from being achieved, particularly with protein containing biological fluids and protein bound compounds.

Citation

ALEXANDER, C. 1996. A study of column switching liquid chromatography in drug analysis. Robert Gordon University, PhD thesis. Hosted on OpenAIR [online]. Available from: https://doi.org/10.48526/rgu-wt-2807271

• Thesis Type: Thesis
• Deposit Date: May 5, 2025
• Publicly Available Date: May 5, 2025
• DOI: https://doi.org/10.48526/rgu-wt-2807271
• Public URL: https://rgu-repository.worktribe.com/output/2807271
• Award Date: Jun 30, 1996