Approaches To High-throughput Quantitative Bioanalysis Using On-line Solid-phase Extraction To Support Pharmacokinetic Study

Pharmacokinetics is the study of the time course of a drug within the body and incorporates the processes of absorption, distribution, metabolism and excretion. To better interpret the effect and toxicity of the drug, an analytical method should be established to determine the drug and its metabolites quantitatively, which helps us to know about their mechanisms of action and their pharmacokinetic properties during the initial state of drug development and in clinical therapy. With the improvement of combinatorial chemistry and separation and purification of natural products, a number of candidate compounds appeared and required pharmacokinetic analysis. Investigators have shown increasing interest in developing and optimizing high-throughput analytical methods with more sensitivity and accuracy for quantitating small molecule drugs, metabolites, and other xenobiotic biomolecules in biological matrices. Based on on-line solid phase extraction and short analytical column, high-throughput bio-analysis method with accuracy and sensitivity was established and utilized in pharmacokinetic studies of new medicine.An automated system using on-line solid-phase extraction and HPLC with UV detection was developed for the determination of faropenem in human plasma and urine. Analytical process was performed isocratically with two reversed-phase columns connected by a switching valve. After simple pretreatment for plasma and urine with acetonitrile, a volume of 100μl upper layer of the plasma or urine samples was injected for on-line SPE column switching HPLC-UV analysis. The analytes were retained on the self-made trap column (Lichrospher C18, 4.6mm×37mm, 25μm) with the loading solvent (20mM NaH2PO4 adjusted pH 3.5) at flow rate of 2ml min^-1, and most matrix materials were removed from the column to waste. After 0.5min washing, the valve was switched to another position so that the target analytes could be eluted from trap column to analytical column in the back-flush mode by the mobile phase (acetonitrile–20mM NaH2PO4 adjusted pH 3.5, 16:84, v/v) at flow rate of 1.5 ml.min^-1, and then separated on the analytical column (UltimateTM XB-C18, 4.6mm×50mm, 5μm). The complete cycle of the on-line SPE preconcentration purification and HPLC separation of the analytes was 5min. Calibration curves with good linearities (r = 0.9994 for plasma sample and r = 0.9988 for urine sample) were obtained in the range 0.02–5μg.ml^-1 in plasma and 0.05–10μg.ml^-1 in urine for faropenem. The optimized method showed good performance in terms of specificity, linearity, detection and quantification limits, precision and accuracy. The method was successfully utilized to quantify faropenem in human plasma and urine to support the clinical pharmacokinetic studies.Another experiment was determination of cefdinir in beagle dog plasma. This experiment contained two sections. Section one, six beagle dogs in a randomized crossover study, divided into three groups, were given a single oral dose of 100mg cefdinir, 100mg hydrosulfate cefdinir sodium and 300mg cefdinir, respectively. Section two, the same six beagle dogs were intravenous injected a single dose of 10mg cefdinir in order to validate the absolute recovery. The blood samples were collected at different time after dosing. All collected blood samples were centrifuged to obtain plasma and the concentration of cefdinir was determined by on-line SPE-HPLC method described as follows. Pharmacokinetics parameter calculations were carried out using non-compartmental analysis method. And these pharmacokinetic parameters would provide reference frame to clinical pharmacokinetic studies.The on-line solid-phase extraction (SPE) high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection was developed for the determination of cefdinir in beagle dog plasma. Plasma samples (100μL) were injected into a C18 SPE column after simple pretreatment with 6% perchloric acid. The analytes were retained on the self-made trap column (Lichrospher C18, 4.6mm×37mm, 25μm) and the biological matrix was washed out with the loading solvent (20mM KH2PO4 adjusted pH3.0) at flow rate of 2 ml·min-1 for 0.2 min. By rotation of the switching valve, cefdinir were eluted in the back-flush mode and transferred to the analytical column(UltimateTM XB-C18, 4.6mm×50mm, 5μm) by the chromatographic mobile phase consisted of methanol-acetonitrile-20mM KH2PO4 (adjusted pH 3.0) (11.25:6.75:82; v/v/v) at flow rate of 1.5 ml·min^-1. The complete cycle of the on-line SPE preconcentration, purification and HPLC separation of the analytes was 4min. The UV detection was performed at 286nm. This method was successfully applied to quantify cefdinir in beagle dog plasma.