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Solid Phase Micro Extraction (SPME) in combination with Gas Chromatography (GC) as a tool for study of the protein binding in human plasma using local anaesthetics of the amide type as model compounds
1999 (English)Independent thesis Advanced level (degree of Master (One Year))Student thesis
Abstract [en]

Solid Phase Micro Extraction, SPME, is a simple sample preparation method for gas chromatography, GC, and liquid chormatography, LC. This extraction technique is fast, easy to handle and requires no solvents. It is also easy to automate and shows good linearity for many analytes. The extraction is based on the partitioning of the analyte between the organic phase on the fused silica fiber and the matrix. Many factors, such as pH, temperature, salts and stirring, affect the equilibrium constant and the equilibrium time. Local anaesthetics are protein bound. Minor changes in the binding capacity can cause severe reactions, e.g., cardiovascular and central nervous side-effects. It is mainly the free, non-protein-bound fraction of local anaesthetics that is responsible for these side-effects. The aim of the present investigation was to evaluate SPME as a tool to determine the protein binding of local anaesthetics of the amide type in human plasma. The influence of pH, temperature and concentration on the protein binding was studied for five substances: Ropivacaine, Prilocaine, Mepivacaine, Bupivacaine and Lidocaine. Solid Phase Micro Extraction (SPME) followed by GC with Nitrogen Phosphor Detector (NPD) was compared with methods described in the literature. This study shows that SPME gives the same protein binding for the five substances as existing methods. The protein binding was investigated at four different concentrations, from 0,5 to 15µM. When the concentration decreased, the protein binding increased. Protein binding of the five substances was also investigated at three different pH-levels, (6,4; 7,4; 9,5). It was found that protein binding increased with increasing pH. The effect of temperature variation (from 32ºC to 40ºC) on protein binding was also investigated. The protein binding was found to decrease with increasing temperature. Ultrafiltrate spiked with each of the substances was used for constructing calibration curves. Good correlation and precision were obtained. Backcalculated control samples gave an accuracy within 20% for all five substances. The acceptance criteria for the validation of the method in this study were within the limits of the international criteria for validation of a method.

Place, publisher, year, edition, pages
1999. , 29 p.
Identifiers
URN: urn:nbn:se:kau:diva-52628Local ID: KEM-9OAI: oai:DiVA.org:kau-52628DiVA: diva2:1101155
Subject / course
Chemistry
Available from: 2017-05-29 Created: 2017-05-29

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