Fully Automated 96-Well Liquid−Liquid Extraction for Analysis of Biological Samples by Liquid Chromatography with Tandem Mass Spectrometry

Abstract

A fully automated high-throughput liquid-liquid extraction (LLE) methodology has been developed for preparation of biological samples using a 96-well LLE plate and a 96-channel robotic liquid handling workstation. The 96-well LLE plate is made of a 96-well filter plate filled with inert diatomaceous earth particles, allowing continuous and efficient extraction of analytes between the aqueous biological sample and the organic extraction solvent. Two carboxylic acid-based protease inhibitor compounds with high and low levels of plasma protein binding were chosen for the development and application of the automated methodology. The LLE extracts of the plasma samples of the two compounds were analyzed by high-performance liquid chromatography with electrospray (ESI) tandem mass spectrometry (LC-MS/MS). The LC-MS/MS method was developed using a rapid gradient LC separation, followed by sample introduction through an ionspray interface in the negative ion mode and tandem mass spectrometric detection with selected reaction monitoring. In the optimized LLE method, a formate buffer solution was first loaded into a 96-well filter plate packed with inert diatomaceous earth material. Then crude plasma samples and a water-immiscible organic solvent, methyl ethyl ketone, were sequentially added to the LLE plate so that LLE would occur in the interface between the two liquid phases on the surface of individual particles in each well. The organic eluate containing extracted analytes was evaporated and reconstituted for LC-MS/MS analysis. This fully automated LLE methodology avoids several disjointed steps involved in a manual or semiautomated LLE method, leading to significantly reduced sample preparation time, increased sample throughput, and clean sample extracts for improved ESI-MS/MS detection. The automated LLE methodology is universal and can be employed for sample preparation of other biological fluids. The complete bioanalytical method, based on the automated LLE and fast gradient LC-MS/MS, was validated and successfully applied to the quantitative analysis of protease inhibitors in rat plasma.

Tandem mass spectrum of a farnesyl transferase inhibitor— gas‐phase rearrangements involving imidazole

“…Recently, electrospray ionization mass spectrometry (ESI/MS) and tandem mass spectrometry (MS/MS), coupled with high performance liquid chromatography, have been used increasingly in many areas of safety and clinical studies. 3,4 Since successful application of these techniques in the areas such as drug metabolism, pharmacokinetics, degradant identification and toxicology depends on a thorough understanding of the ESI/MS and MS/MS data of the compounds tested, the MS/MS data of protonated 1 are analyzed in detail in this report. As part of a continuous effort to analyze MS/MS data of novel drugs, 5 -7 this work reveals some unique features of the MS/MS spectra of this important class of farnesyl protein transferase inhibitors.…”

Tandem mass spectrum of a growth hormone secretagogue: Amide bond cleavage and resultant gas-phase rearrangement

“…Recently, electrospray ionization mass spectrometry (ESI/MS) and tandem mass spectrometry (MS n ) have been applied to many areas of drug discovery and development [2,3]. The successful application of these techniques requires a thorough understanding of the ESI/MS and MS n data of the drugs under investigation.…”

A review of current trends and advances in modern bio-analytical methods: Chromatography and sample preparation

“…Peng et al reported a fully automated high-throughput LLE method for the preparation of biological samples using a 96-well plate and 96-channel robotic liquid-handling workstation in order to completely automate the entire process. Extraction time was substantially shortened using this approach [130].…”