Approaches To Bioanalysis Using Automated Online Solid Phase Extraction-High Performance Liquid Chromatography

Sample pretreatment and sample preparations are critical steps in bioanalysis of active pharmaceutical principals in in vivo study. It is estimated that about80%of the efforts and research funding have been spent on the sample pretreatment and sample preparation steps in pharmacokinetic (PK) study. The objectivs of this research is focused on automation of sample pretreatment and sample preparation to enable accurate, sensitive and high-throughput bioanalytical methods for PK study by application of on-line solid phase extraction (SPE) technology.First of all, a fully automated online SPE coupled with high-performance liquid chromatography (HPLC) with diode array detection (DAD) method was developed for determination of bavachinin in mouse plasma. Analytical process was performed on two reversed-phase columns (Capcell MF Ph-1cartridge&analytical column) connected via a Valco6-port switching valve. The lowest limit of quantification (LLOQ) of the assay was20ng/ml. The established automated online SPE-HPLC-DAD method demonstrated good performance in terms of linearity, specificity, detection and quantification limits, precision and accuracy. It was successfully utilized to quantify bavachinin in mouse plasma to enable PK study. The PK properties of bavachinin were characterized as rapid oral absorption, high clearance, and poor absolute bioavailability.Optimization of multiparameters in SPE-HPLC bioanalytical system is a challenging task. Response surface methodology (RSM) was utilized for rapid and systematic optimization of on-line SPE parameters to maximize the response and separation of WM-5. The optimization was performed with Box-Behnken designs. Four major parameters were investigated for their contributions to the response and separation of WM-5, with a total of29experiments being performed for each instrument, respectively. Quantitative determination of WM-5in mouse plasma was performed to evaluate the statistical significance of the parameters on chromatographic response. A fully automated online SPE-HPLC-DAD method was developed for the determination of WM-5in mouse plasma. The single run was within8min, and LLOQ of WM-5was20ng/ml. The optimized method demonstrated good performance in terms of specificity, LLOQ, linearity, recovery, precision and accuracy.A fully automated online solid phase extraction coupled to high-performance liquid chromatography-tandem mass spectrometry (online SPE-HPLC-MS/MS) method was described for the simultaneous determination of a variety of cardiovascular drugs in rat plasma. Following a simple centrifugation step, a10μl aliquot of the plasma sample was injected directly onto the HPLC system. The LiChrospher(?) RP-18ADS cartridge was chosen as the online SPE cartridge, the analytes were quantified by measurement of an API4000+triple-quadrupole tandem mass spectrometer in multiple-reaction monitoring mode. The procedure was easier to execute and required significantly less sample handling than methods currently described in the literature. The correlation study of the physicochemical parameters and the analytical parameters of analytes may provide a solid basis for SPE cartridge selection and method optimization for the therapeutic drug monitoring and PK study of cardiovascular drugs.Nucleosides are a class of chemical structrues having widely clinical applications. However, the bioanalysis of nucleosides is very challenging due to their high polarity and high solubility. The matrix clearance and the extraction of target analytes from the matrix samples in in vivo PK study have attracted great attention in bioanalytical research. In this study, online SPE technology has been applied to address the difficulities in sample pretreatment and sample preparation of nucleosides in human plasma. A fully automated online SPE-HPLC-MS/MS method was established for the simultaneous determination of a variety of nucleoside reverse transcriptase inhibitors (Lamivudine, Zidovudine, Didanosine and Emtricitabine) in human plasma. After injection of10μl aliquot of the plasma sample onto the online SPE-HPLC-MS/MS system, the Oasis (?)MCX cartridge was performed as the online SPE cartridge, the analytes were quantified by measurement of an API4000+triple-quadrupole tandem mass spectrometer in multiple-reaction monitoring mode, and the analysis was finished within10min with no significant matrix effect. The procedure was easier to execute and required less sample handling than methods currently described in the literature. The established automated online SPE-HPLC-MS/MS method demonstrated good performance in terms of linearity, specificity, detection and quantification limits, precision, accuracy, recovery and matrix effect, which were within the criteria of FDA requirement of biological sample analysis. Moreover, a fully automated online SPE-HPLC-MS/MS method was established for the simultaneous determination of Ribavirin and Tribavirin in human plasma based on their physical and chemical properties. Because of the high polarity of Ribavirin and Tribavirin, a Bond Elut Phenylboronic acid SPE cartridge was developed and used in this study. Through optimization of chromatography and mass spectrometry, Ribavirin and Taribavirin were trapped in the SPE cartridge successfully, and thereafter they were eluted by acidic mobile phase. The precipitation of plasma proteins was effectively avoided by using low proportion of organic mobile phase. In conclusion, the procedure was easier to execute and required less sample handling than methods currently described in the literature. The established automated online SPE HPLC-MS/MS method demonstrated good performance in terms of linearity, specificity, detection and quantification limits, precision, accuracy, recovery, and matrix effect.