High-performance liquid chromatographic method for the simultaneous determination of pentisomide and its major metabolite N-desisopropylpentisomide in plasma, urine and tissues using solid-phase extraction.
A rapid, sensitive and selective high-performance liquid chromatographic method for the simultaneous determination of pentisomide and its major metabolite desisopropylpentisomide in plasma, urine and tissues has been developed. The method for plasma samples, urine samples and tissue samples, after homogenizing with 50% ethanol, involves extraction of samples via activated Bond-Elut C8 disposable columns with methanol at pH 10 after addition of internal standard, and initially on column washing of samples at pH 10 with water and acetonitrile. The obtained methanolic extract is evaporated to dryness under nitrogen at 25 degrees C; the sample residue is then reconstituted in mobile phase and an aliquot of this solution is injected into the liquid chromatograph. Separation is performed using a Nova-Pak C18 4 microns particle size column operating in combination with radial compression separation unit and a methanol-water-di-sec.-butylamine-phosphoric acid (40:60:0.5:0.2, v/v) pH 3.5 mobile phase with ultraviolet detection at 258 nm. Endogenous substances or a variety of drugs concomitantly used in pentisomide therapy, with the exception of disopyramide, do not interfere with the assay. The mean recovery of pentisomide and desisopropylpentisomide from plasma and urine and from tissues is more than 91 and 86%, respectively. The limit of detection of the assay is 10 ng/ml for both drugs. The intra- and inter-day coefficient of variation for replicate analyses of spiked plasma samples is less than 7 and 8%, respectively. Mean steady-state plasma levels of pentisomide and desisopropylpentisomide in patients on chronic oral therapy are reported.
Selected Solid Phase Extraction Applications in Drug Analysis
The following chapters will outline specific analytical methods that have been published or have been developed in the laboratory for selected drugs, highlighting the use of solid phase extraction.
Protein binding of propisomide.
This paper describes the protein binding of propisomide to human serum and isolated proteins using equilibrium dialysis. The drug is exclusively bound to alpha1-acid glycoprotein with high affinity. The binding is saturable even at low concentrations of the drug. Thus, the fraction unbound varied from 0.05 to 0.60 with decreasing serum concentration. The major metabolite of the drug or other drugs with affinity for alpha1-acid glycoprotein can displace propisomide from its binding site only when present in serum at high levels. Two ultrafiltration techniques are compared to equilibrium dialysis for the determination of serum protein binding of propisomide. Ultrafiltration does not give reliable results. Equilibrium dialysis is retained as an accurate method for the determination of the fraction unbound of propisomide.