Addressing The Challenge of Limited Sample Volumes in In Vitro Studies with Capillary-Scale Microfluidic LC–MS/MS

Abstract

Miniaturization of chromatographic separation systems provides a means of greatly increasing sensitivity in LC-MS. In this article, we demonstrate the use of an integrated microfluidic chromatographic device for the LC-MS/MS investigation of the in vitro microsomal metabolism of the model drug propranolol using a sample volume of 1 µl of a 1-µM incubation. With such samples the system was capable of obtaining high-quality MS and MS/MS data from the injection of test drug substance containing sufficient information to correctly derive the structure of the drug metabolites. The analytical column was tolerant to the injection of a large percentage of organic solvent in the sample and still delivered a high-quality separation. The data suggest that these types of micro-LC-MS/MS devices are robust enough for routine applications and well suited to the analysis of small samples. Other potential applications include the generation of pharmacokinetic profiles from the reduced sample volumes obtained from serially bled small rodent studies, or the facilitation of analysis of limited-volume samples from neurological studies.

Advances in liquid chromatography coupled to mass spectrometry for metabolic phenotyping

“…Miniaturization of the chromatographic system to enable it to cope with reduced sample size does not represent a particular problem as it is a scalable process. Recent developments in microscale LC mean that it is now beginning to see applications in small molecule analysis, such as drug bioanalysis and metabolite identification [65] with chromatographic performance similar to conventional scale LC. In addition, significant improvements in MS response, in some cases 20-fold, have been noted using these microseparation approaches [65].…”

“…Recent developments in microscale LC mean that it is now beginning to see applications in small molecule analysis, such as drug bioanalysis and metabolite identification [65] with chromatographic performance similar to conventional scale LC. In addition, significant improvements in MS response, in some cases 20-fold, have been noted using these microseparation approaches [65]. Applications of micro (or nano) LC-MS to metabolite profiling include the determination of polar cationic metabolites in human cerebrospinal fluid (CSF) [66], and polar anionic metabolites in HeLa cells, mouse-brain extracts, plasma and CSF [67].…”

Practical Applications of Integrated Microfluidics for Peptide Quantification

“…The ability to reuse these animals facilitates the investigation of disease models. In addition, if the sample volume required can be sufficiently minimized, a full PK profile can be obtained from a single animal in contrast to the previous practice of obtaining composite profiles whereby each time point was represented by an individual animal [2]. This ultimately future science group Research Article Chambers, Lame, Rainville et al improves the quality and reproducibility of PK data obtained from rodent models.…”

“…In theory, if the particle size and column length are kept constant, reducing the column diameter should significantly increase sensitivity if the same sample volume is injected. For concentration sensitive detectors, such as UV, this increase in sensitivity is directly related to the ratio of the square of the two column diameters, that is, d 1 2 /d 2 2 where d 1 is the diameter of the larger column and d 2 is the diameter of the smaller column. Although historically, MS was thought to be a concentration-dependent detector [5,6], this has recently been disproven [7,8].…”

“…Microsampling has emerged as an attractive option to traditional methods [1][2][3] for several reasons. Smaller rodent animal models (i.e., mice versus rats) are being used and there is a strong desire to reduce overall animal usage both from a financial and humanitarian perspective.…”