Highly sensitive LC-MS-MS analysis of a pharmaceutical compound in human plasma using monolithic phase-based on-line extraction.

Monolithic columns have been used in recent years for fast chromatographic separation due to their high permeability and low backpressure. We have explored the potential of monolithic material as sample preparation tool in bioanalytical applications. By taking advantage of monolithic columns' online concentration capability, we have developed a highly sensitive liquid chromatography-tandem mass spectrometry assay for quantitative determination of a pharmaceutical compound in human plasma. The assay was fully validated to satisfy the requirements of precision and accuracy, selectivity, matrix effect, and reproducibility. A linear dynamic range from 0.011 ng/mL to 12.3 ng/mL was established as the calibration standard. The percentage of bias for quality control samples was between -9.9% and -2.5%. Coefficient of variation, a measurement of precision, was within 9.9%. On-line extraction with monolithic support provided adequate sample cleanup and on-line concentration of the analyte. The assay exhibited good tolerance to matrix effect and has been applied successfully to a clinical study. The incurred sample analysis showed that original and repeat values were within +/-10.1% for all assay samples.

Bioanalytical hydrophilic interaction chromatography: recent challenges, solutions and applications.

Hydrophilic interaction chromatography (HILIC) has, in recent years, been shown to be an important supplement to reversed-phase liquid chromatography for polar analytes. HILIC, in conjunction with tandem mass spectrometry (MS/MS), has been steadily gaining acceptance in the analysis of polar compounds from complex biological matrices. This hyphenated technique offers the advantages of improved sensitivity by employing high organic content in the mobile phase, shortened sample preparation time with direct injection of the organic-solvent extracts of biological samples and the potential for ultra-fast analysis because of low-column backpressure. This article reviews recent challenges presented by HILIC, advancements in the better understanding of retention characteristics of analytes with different mobile- and stationary-phase compositions and solutions to ion suppression and interference problems encountered in HILIC-MS/MS assays. Applications of HILIC-MS/MS are summarized, including those for pharmacokinetic studies, metabolic studies, therapeutic drug monitoring and clinical diagnostics.

Simultaneous LC-MS/MS quantitation of a highly hydrophobic pharmaceutical compound and its metabolite in urine using online monolithic phase-based extraction.

This article describes the use of monolithic material as extraction support in simultaneous LC-MS/MS quantitation of a highly hydrophobic pharmaceutical compound and its hydroxylated metabolite in urinary matrix. DMSO was added to urine samples to aid the solubilization of analytes during storage and transfer. Samples were then diluted and injected onto an online extraction system for high-throughput analysis in an automated fashion. A linear range of 1.03-103 ng/mL was validated for the parent compound and 7.02-1400 ng/mL for the metabolite. The accuracy (%bias) at the lower LOQ (LLOQ) for the parent compound was -2.9% and the precision (%CV) at the LLOQ was 5.4%, while the accuracy at LLOQ for the metabolite was 6.3% and the precision at LLOQ was 5.5%. The results show that quantitative LC-MS/MS analysis by online extraction using the monolithic support is reproducible, sensitive, and accurate enough to satisfy bioanalytical testing requirements in a good laboratory practice (GLP) regulated environment. Monolithic phase based online extraction provided efficient sample cleanup, which was evidenced in matrix effect testing against multiple lots of urinary matrix. The method described within can be a generic approach for quantitative analysis of analytes with poor solubility in aqueous matrix.

Recent advances in high-throughput quantitative bioanalysis by LC-MS/MS.

Liquid chromatography linked to tandem mass spectrometry (LC-MS/MS) has played an important role in pharmacokinetics and metabolism studies at various drug development stages since its introduction to the pharmaceutical industry. This article reviews the most recent advances in sample preparation, separation, and the mass spectrometric aspects of high-throughput quantitative bioanalysis of drug and metabolites in biological matrices. Newly introduced techniques such as ultra-performance liquid chromatography with small particles (sub-2 microm) and monolithic chromatography offer improvements in speed, resolution and sensitivity compared to conventional chromatographic techniques. Hydrophilic interaction chromatography (HILIC) on silica columns with low aqueous/high organic mobile phase is emerging as a valuable supplement to the reversed-phase LC-MS/MS. Sample preparation formatted to 96-well plates has allowed for semi-automation of off-line sample preparation techniques, significantly impacting throughput. On-line solid-phase extraction (SPE) utilizing column-switching techniques is rapidly gaining acceptance in bioanalytical applications to reduce both time and labor required to produce bioanalytical results. Extraction sorbents for on-line SPE extend to an array of media including large particles for turbulent flow chromatography, restricted access materials (RAM), monolithic materials, and disposable cartridges utilizing traditional packings such as those used in Spark Holland systems. In the end, this paper also discusses recent studies of matrix effect in LC-MS/MS analysis and how to reduce/eliminate matrix effect in method development and validation.