Approaches To High-throughput Quantitative Bioanalysis Using Liquid Chromatography 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 help 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 fast HPLC using a monolithic silica column, sample preparation in 96-well format and liquid chromatography tandem mass spectrometry (LC-MS/MS), high-throughput bio-analysis method with accuracy and sensitivity was established and utilized in pharmacokinetic studies of new medicine.The development of the conventional particle-based silica column and the rapid and sensitive monolithic silica column-based methods for determination of the active metabolite NM394 of prulifloxacin in human plasma and a new antifungal drug ADKZ in rat and dog plasma were reported. It was investigated if the separation on the monolithic column was suitable when it was transferred from conventional particle-based silica column. Different ratios of the mobile phase, pH value as well as column temperatures and flow rate were studied in order to shorten the retention time and to avoid the interference of endogenous substances. The retention of analytes on the monolithic column was similar to it on the conventional column. The very high porosity of the stationary phase allowed chromatography with much lower back-pressure than on conventional column, and therefore, flow rates up to 2 ml/min shortened the analysis time to increase the throughput of the assay. One-step protein precipitation with 10% perchloric acid on a 200μl plasma sample was used. The separation was performed at ambient temperature on a Chromolith RP-18e column (4.6×100mm) using an eluent of acetonitrile-water adjusted to pH 3.0 with phosphoric acid (15:85,v/v), at a flow rate of 2 ml/min. NM394 and the internal standard (IS), lomefloxacin were monitored atλ_ex of 280 nm ,λ_em of 425 nm. The retention time of NM394 and IS was 3.0 and 2.3 min, respectively and the total run time was 4 min, less than the former assay.The dog plasma sample was extracted by n-Hexane after the addition of sodium hydroxide solution and separated by a Chromolith RP-18e column (4.6×100mm) with UV detection at 230 nm. The mobile phase consisted of acetonitrile-0.02M sodium dihydrogen phosphate adjusted to pH 3.8 (35:65,v/v) and was delivered at a flow rate of 2ml/min. The retention time for ADKZ was 2.7 min and IS, 0522 3.2 min. Compared with the former method, the assay run time decreased from 10 min to 4 min and the lower limit of quantification was 10 ng/ml.A simultaneous solid phase extraction with a mixed-mode column was used to extract the calcium-channel antagonists amlodipine and the angiotensin-Ⅱantagonists irbesartan from the beagle dog plasma. The separation was performed on a Chromolith RP-18e column (4.6×100mm) using mobile phase of acetonitrile-methnal-0.02M sodium dihydrogen phosphate adjusted to pH 3.0 (25:20:55,v/v/v) to achieve an entire separation of the two drugs and two internal standards in less than 6.5 min. The proposed technique has been applied in the pharmacokinetic study in beagle dogs after co-administration of amlodipine and irbesartan and especially was suitable for routine drug monitoring of resistant hypertensive patients.The development and full validation of the rapid and sensitive LC-MS/MS-based method focused on improvement of sample preparation in 96-well format, matrix effect, cross-talk and carry-over to ensure the accuracy and sensitivity of the assay.We have demonstrated a method for determination of fudosteine in human plasma by a rapid LC-MS/MS analysis. The simple and fast protein precipitation in 96-well format for sample preparation prior to LC-MS/MS reduced sample preparation time, increased sample throughput and only a minimum of 50μL aliquots of plasma was required. Despite of the presence of matrix effects, the present LC-MS/MS method was reliable because using the structure analogue, erdosteine as the IS could tracked the fudosteine well in the mass spectrometer ion-source for their similar ionization suppression. Validation was successfully performed: selectivity, sensitivity, precision, accuracy, recovery, and calibration curves fulfilled analytical validation criteria, which meet the purpose of the pharmacokinetic study. The simplicity and rapidness of the method, using 96-well protein precipitation and sample turnover rate of 3.0 min per sample, make it an attractive procedure in high-throughput bioanalysis of fudosteine.Clenbuterol was extracted from dog plasma samples by liquid-liquid extraction in 96-well format. Extraction solvents were compared according to the results how less the matrix effect would be and how much the extraction efficiency. The extraction efficiency was stable and the ion suppression was relieved when the plasma sample was diluted. The addition of 0.01% formic acid in mobile phase and the wash solution had aided in elimination or minimization of carry-over for clenbuterol in autosampler, which ensured the linearity of the calibration curve within the range of 0.1～10 ng/ml. The method was successfully applied to the bioequivalence study of extended action tablet of clenbuterol hydrochloride.Phakellistatin 13 (PK13), a new cyclic heptapeptide isolated from the sponge Phakellia fusca Thiele, showed good anti-tumor activity. In order to address the pharmacokinetics of PK13 in rats,a we employed a sensitivity and high-throughput methodology for determination of PK13 in rat plasma. PK13 was extracted from plasma by Sirocco protein precipitation plate followed by LC-MS/MS analysis. The described assay reduced ion suppression and improved the extraction efficiency. The lower limits of quantification of 0.1 ng/ml was obtained in 50μl rat plasma . the method was successfully applied in measuring levels of PK13 in rat plasma following intravenous administration of 20, 50 and 100μg/Kg of peptide in rat, which was suitable for the concessive preclinical pharmacokinetic studies on PK13.