Abstract
The importance of drug analysis in pharmaceutical and biomedical laboratories has led to the development of increasingly sophisticated methods of analysis. This has resulted in improved sample preparation procedures to meet the specific requirements of the analytical instruments. Selective sample preparation has been the focus of attention in recent years, particularly in chromatographic analysis. The development of immunoaffinity solid phase extraction (SPE) for biological fluids was one of the main objectives of this study. Using the model compounds clenbuterol and morphine, immunoaffinity SPE was optimised for application to biological fluids. The optimisation of the immunoaffinity placed emphasis on the loading of samples into columns, washing to remove unbound materials and the elution of drugs from the column in the minimum volume possible. The antibody-drug interaction, mainly due to hydrophobic and ionic interaction was indicated by the use of low pH elution buffers in association with polarity reducing agents (organic modifier). The extraction efficiency of the immunoaffinity extraction was >98%. The immunoextraction procedure for both model compounds was optimised for elution in a single 1 ml elution fraction which was analysed directly by high performance liquid chromatography. High column capacity was desirable in the immunoaffinity column as this made the column reusable. Precipitating the plasma protein greatly improved the precision and accuracy of the method. The method coefficient of variation (%CV) for plasma clenbuterol after protein precipitation was < 5% for both within and between-day analyses. For urine morphine, the within-day precision was high (%CV between 13-24) for all concentration levels used (50, 250 and 450 ng/ml). The between-day precision was within the method validation acceptable limits. Preconcentration of drugs was effective using the immunoaffinity SPE. Samples diluted up to 100-fold were able to preconcentrate analytes with highly efficient recoveries. Immunoextraction using vacuum resulted in poor recoveries at flow rates above 4 ml/min. Molecularly imprinted polymers (MIP) were also evaluated as a selective SPE sorbent. Tamoxifen MIP was prepared using a simple laboratory set-up. Tamoxifen templates were removed from the MIP sorbents by methanol/acetic acid (4:1). The optimisation of the MIP showed acetonitrile to be suitable as a washing solvent and acetonitrile/acetic (4:1) as an elution solvent. A standard curve of tamoxifen in acetonitrile was linear with r=97%. However, MIP demonstrated different levels of affinity to morphine, chlortoluron, clenbuterol and oxyprenolol. This may be due to the non-specific binding of the drugs to the tamoxifen MIP sorbents. Tamoxifen in urine and plasma was successfully extracted by the tamoxifen MIP. The study indicated that immunoaffinity and MIP sorbent have excellent potential as a selective SPE resulting in high extraction efficiency and clean chromatographic tracing. However, further studies and optimisation are necessary before both methods could replace the present sample preparation procedures such as SPE and solvent extraction in a routine laboratoiy.