Students

Name: Wong Kwok-Kong

Post: Full-time M.Phil

Enrolled: 1993-1995

Origin: Mainland China

Supervisor: Prof. Tony Gin

Title of Thesis:

Propofol: Analytical Techniques and Applied Pharmacokinetics

Outline of the Thesis:

Propofol is a commercially available intravenous anaesthetic agent. The main feature of propofol is rapid recovery from anaesthesia, whether given as a bolus for induction of anaesthesia or by infusion to maintain anaesthesia.

The aim of this research was to develop analytical techniques for measuring the concentration of propofol. It details the assay of propofol in blood samples using high-pressure liquid chromatography (HPLC) with fluorescence detection, and propofol protein binding studies using equilibrium dialysis. Examples are given of clinical applications of these techniques.

The reversed phase HPLC method used was simple, sensitive, and reliable. The assay involved a single extraction of the drug and internal standard, thymol, from blood buffered with 0.1 M sodium dihydrogen phosphate buffer into cyclohexane. The organic extract, basified with tetramethylammonium hydroxide, was evaporated to dryness at 37°C under nitrogen. The residue was redissolved in 80 µl acetonitrile and an aliquot of the concentrate was injected into a C18 reversed-phase column. The mobile phase consisted of 66% (v/v) acetonitrile in Milli-Q water containing 1% v/v acetic acid and was eluted at 1.7 ml/min. The components of the column effluent was monitored by a fluorometric detector with excitation and emission wavelengths set at 276 nm and 310 nm, respectively. The main advantage of my method for detecting propofol is that only 0.5 ml of whole blood is required. This facilitates pharmacokinetic studies of propofol in children. The calibration graphs were linear over the range 2-3000 ng/ml with coefficients of variation ranging from 0.38 to 7.86%, while the limit of detection was approximately 2 ng/ml. The intraassay coefficient of variation of propofol in whole blood was 6.24% at 100 ng/ml and 6.10% at 500 ng/ml, while the interassay coefficient of variation was 3.18% at 100 ng/ml and 1.81% at 500 ng/ml. The extraction recovery results obtained for propofol in whole blood are 104% at 100 ng /ml and 99% at 500 ng/ml. Stability of propofol in whole blood is one month. The results of propofol assay in plasma are similar to propofol assay in whole blood.

For the protein binding assay, equilibrium dialysis was performed using a Spectrum equilibrium Dialyser. Drug-containing plasma samples or protein solutions (1 ml) were dialysed against drug-free Sorensen's phosphate buffer (1 ml; pH 7.4) in teflon dialysis chambers separated by Spectra/Por dialysis membrane. The effect of drug concentration on protein binding was determined within the range of 0.25-3.00 µg/ml of propofol. The teflon cells rotate at 15 rpm at 37°C. The optimum dialysis time was 240 min. Propofol concentration in samples were assayed by HPLC as described above. The intraassay coefficient of variation of propofol protein binding was 0.11% (3.65% Free), while the interassay coefficient of variation of propofol protein binding was 0.23% (8.57% Free). The extraction recovery results obtained for propofol in plasma are 96.42% in unheated samples, 90.64% for samples heated at 37°C, and 97.15% samples dialysed at 37°C in a water bath.

One application of my research was the prospective testing in chinese children of a previously published pharmacokinetic model driven algorithm for computer controlled infusion of propofol. The model was revised using an iterative linear least squares regression and minimizing total mean squared prediction error for each patient. The precision of the revised model was 21.08% and bias was 0.19%.

Another application of my research was to investigate protein binding of propofol in different populations because pharmacological response to drugs is influenced by the free fraction of drug. There were no differences in protein binding between young children [< 3 years, 98.77 ± 0.29% (mean ± S.D.)], children (3-12 years, 98.69 ± 0.33%) and adults (98.35 ± 0.29%).

Pregnant patients had increased free propofol, 1.41 ± 0.30% compared with nonpregnant patients, 1.08 ± 0.26% (p=0.004). Free propofol in umbilical plasma 3.40 ± 0.61% was greater than that in maternal (pregnant) plasma (p=0.001). These findings indicate that further simultaneous pharmacodynamic/pharmacokinetic studies would be useful to determine the clinical significance of changes in protein binding.