Evaluation of solid-phase microextraction for the study of protein binding in human plasma samples

Solid-phase microextraction (SPME) in combination with capillary gas chromatography and a nitrogen-phosphorous detector is used to study protein binding in human plasma samples. Local anesthetics of the amide-type (ropivacaine, bupivacaine, mepivacaine, prilocaine, and lidocaine) are used as model compounds in this evaluation. Carbowax/divinylbenzene (CW/DVB), polyacrylate, and polydimethylsiloxane fibers are tested. Sampling on CW/DVB fibers give the highest recovery in plasma samples compared with other fibers. Ultrafiltrate spiked with each of the substances is used for the construction of calibration curves. The protein binding is investigated at four different total concentrations from 0.5 to 15.0 microM. The degree of protein binding increases when the solute concentration decreases. Protein binding of the five solutes is investigated at four pH levels (6.4, 7.4, 8.4, and 9.4). It is found that protein binding increased with increasing pH. The influence of temperature variation (from 32 degrees C to 40 degrees C) on protein binding is also investigated. The protein binding decreases when the temperature increases. The methodology is validated and good correlation and precision are obtained. Back-calculated quality control samples give accuracy within 20% of theoretical values for all five substances. This study shows that SPME as a sample-preparation method gives the same protein binding for the studied local anesthetics as that achieved using earlier presented methods.